U.S. patent application number 14/925346 was filed with the patent office on 2017-08-24 for method of producing erythrocytes.
This patent application is currently assigned to Anthrogenesis Corporation. The applicant listed for this patent is Anthrogenesis Corporation. Invention is credited to Stewart Abbot, Mohammad A. Heidaran, Lin Kang, Vanessa Voskinarian-Berse, Andrew Zeitlin, Xiaokui Zhang.
Application Number | 20170240860 14/925346 |
Document ID | / |
Family ID | 54609135 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170240860 |
Kind Code |
A1 |
Heidaran; Mohammad A. ; et
al. |
August 24, 2017 |
METHOD OF PRODUCING ERYTHROCYTES
Abstract
Provided herein are methods of producing erythrocytes from
hematopoietic cells, particularly hematopoietic cells from
placental perfusate in combination with hematopoietic cells from
umbilical cord blood, wherein the method results in accelerated
expansion and differentiation of the hematopoietic cells to more
efficiently produce administrable erythrocytes. Further provided
herein is a bioreactor in which hematopoietic cell expansion and
differentiation takes place.
Inventors: |
Heidaran; Mohammad A.;
(Chatham, NJ) ; Zhang; Xiaokui; (Livingston,
NJ) ; Kang; Lin; (Edison, NJ) ; Zeitlin;
Andrew; (Basking Ridge, NJ) ; Voskinarian-Berse;
Vanessa; (Millington, NJ) ; Abbot; Stewart;
(Warren, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Anthrogenesis Corporation |
Warren |
NJ |
US |
|
|
Assignee: |
Anthrogenesis Corporation
Warren
NJ
|
Family ID: |
54609135 |
Appl. No.: |
14/925346 |
Filed: |
October 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12187337 |
Aug 6, 2008 |
9200253 |
|
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14925346 |
|
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60963894 |
Aug 6, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/105 20130101;
C12N 2501/11 20130101; C12N 2501/145 20130101; C12N 5/0641
20130101; C12N 2501/22 20130101; C12N 2501/999 20130101; C12N
2501/26 20130101; C12N 2501/2306 20130101; C12N 2501/125 20130101;
C12N 2501/2311 20130101; C12N 2501/135 20130101; C12N 2500/99
20130101; C12N 2501/2303 20130101; C12N 2501/14 20130101 |
International
Class: |
C12N 5/078 20060101
C12N005/078 |
Claims
1. A method of producing erythrocytes, comprising (a) expanding a
plurality of hematopoietic cells in the absence of feeder cells,
optionally in contact with an immunomodulatory compound, wherein
the immunomodulatory compound increases the number of hematopoietic
cells compared to a plurality of hematopoietic cells not in contact
with the immunomodulatory compound, to produce a first expanded
hematopoietic cell population; (b) expanding the first expanded
hematopoietic cell population in the presence of a plurality of
feeder cells to produce a second expanded hematopoietic cell
population; (c) contacting said second expanded hematopoietic cell
population with one or more factors that cause differentiation of
hematopoietic cells in said second expanded hematopoietic cell
population into erythrocytes; and (d) isolating said erythrocytes
from said second expanded hematopoietic cell population.
2. The method of claim 1, wherein said hematopoietic cells are
CD34.sup.+.
3. The method of claim 1, wherein said hematopoietic cells are
Thy-1.sup.+, CXCR4.sup.+, CD133.sup.+ or KDR.sup.+.
4. The method of claim 2, wherein said hematopoietic cells are
CD45.sup.-.
5. The method of claim 1, wherein said hematopoietic cells are
HLA-DR.sup.-, CD71.sup.-, CD2.sup.-, CD3.sup.-, CD11b.sup.-,
CD11c.sup.-, CD14.sup.-, CD16.sup.-, CD24.sup.-, CD56.sup.-,
CD66b.sup.- and/or glycophorin A.sup.-.
6. The method of claim 1, wherein said hematopoietic cells are
obtained from cord blood, placental blood, peripheral blood, or
bone marrow.
7. The method of claim 1, wherein said hematopoietic cells are
obtained from placental perfusate.
8. The method of claim 1, wherein said hematopoietic cells are
obtained from umbilical cord blood and placental perfusate.
9. The method of claim 6, wherein said placental perfusate is
obtained by passage of perfusion solution through only the
vasculature of a placenta.
10. The method of claim 7, wherein said placental perfusate is
obtained by passage of perfusion solution through only the
vasculature of a placenta.
11. The method of claim 1, wherein said hematopoietic cells are
human hematopoietic cells.
12. The method of claim 1, wherein said feeder cells are from the
same individual as said hematopoietic cells.
13. The method of claim 1, wherein said feeder cells are from a
different individual than said hematopoietic cells.
14. The method of claim 12, wherein said feeder cells are adherent
placental stem cells, bone marrow-derived mesenchymal stem cells,
mesenchymal stem cells from peripheral blood, mesenchymal stem
cells from cord blood, or stromal stem cells, or a combination of
any of the foregoing.
15. The method of claim 12, wherein said feeder cells are adherent
placental stem cells.
16. The method of claim 15, wherein said adherent placental stem
cells are: CD200.sup.+ or HLA-G.sup.+; CD73.sup.+, CD105.sup.+, and
CD200.sup.+; CD200.sup.+ and OCT-4.sup.+; CD73.sup.+, CD105.sup.+
and HLA-G.sup.+; CD73.sup.+ and CD105.sup.+ and facilitate the
formation of one or more embryoid-like bodies in a population of
isolated placental cells comprising said stem cells when said
population is cultured under conditions that allow formation of
embryoid-like bodies; OCT-4.sup.+ and which facilitate formation of
one or more embryoid-like bodies in a population of isolated
placental cells comprising said stem cell when cultured under
conditions that allow formation of embryoid-like bodies;
CD10.sup.+, CD34.sup.-, CD105.sup.+, and CD200.sup.+;
HLA-A,B,C.sup.+, CD45.sup.-, CD133.sup.- and CD34.sup.-;
CD10.sup.+, CD13.sup.+, CD33.sup.+, CD45.sup.-, CD117.sup.- and
CD133.sup.-; CD10.sup.-, CD33.sup.-, CD44.sup.+, CD45.sup.-, and
CD117.sup.-; HLA A,B,C.sup.+, CD45.sup.-, CD34.sup.-, CD133.sup.-;
positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and/or
HLA-G, and/or negative for CD117; CD200.sup.+ and CD10.sup.+, as
determined by antibody binding, and CD117.sup.-, as determined by
both antibody binding and RT-PCR; or CD10.sup.+, CD29.sup.-,
CD54.sup.+, CD200.sup.+, HLA-G.sup.+, HLA class I.sup.+ and
.beta.-2-microglobulin.sup.+.
17. The method of claim 1, wherein a plurality of said
hematopoietic cells is blood type A, blood type O, blood type AB,
blood type O; is Rh positive or Rh negative; blood type M, blood
type N, blood type S, or blood type s; blood type P1; blood type
Lua, blood type Lub, or blood type Lu(a); blood type K (Kell), k
(cellano), Kpa, Kpb, K(a+), Kp(a-b-) or K-k-Kp(a-b-); blood type
Le(a-b-), Le(a+b-) or Le(a-b+); blood type Fy a, Fy b or Fy(a-b-);
or blood type Jk(a-b-), Jk(a+b-), Jk(a-b+) or Jk(a+b+).
18. The method of claim 17, wherein the hematopoietic cells are
type O, Rh positive; type O, Rh negative; type A, Rh positive; type
A, Rh negative; type B, Rh positive; type B, Rh negative; type AB,
Rh positive or type AB, Rh negative.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 12/187,337, filed Aug. 6, 2008, now U.S. Pat. No. 9,200,253,
which claims the benefit of U.S. Provisional Application No.
60/963,894, filed Aug. 6, 2007, each of which is incorporated by
reference herein in its entirety.
1. FIELD
[0002] Provided herein, generally, are methods of expanding
hematopoietic cell populations, e.g., CD34.sup.+ cell populations,
and methods of producing administrable units of erythrocytes from
such cell populations. Also provided herein is a bioreactor that
accomplishes such expansion and differentiation.
2. BACKGROUND
[0003] Each year in the United States approximately 13 million
units of blood are used for transfusion or to generate life-saving
blood products such as platelets. Voluntary blood donation is
utilized by the Red Cross and other agencies to procure from about
500 mL to about 1000 mL whole blood samples. Self-screening of
voluntary donation is relatively safe and effective in the US and
Western Europe where the incidences of HIV and other adventitious
pathogens are relatively low. However, in countries in which HIV
and hepatitis are endemic, procurement of safe blood for
transfusion can be highly problematic. As an alternative to
voluntary blood donation many groups have attempted to develop safe
artificial blood substitutes that could undergo long-term storage.
While some of these products show significant promise in
transiently treating traumatic blood loss, such products are not
designed for long-term substitution of red blood cell function.
Increasingly there is a need to develop a safe and plentiful supply
of erythrocytes that can be administered to patients on the
battlefield or civilian hospital settings around the world.
[0004] Conventional methods for producing erythrocytes are either
inefficient, too small in scale, or too laborious to allow for the
continuous, on-site production of erythrocytes. Conventional dish
or flask-based culture systems are associated with discontinuous
medium exchange, and generally dish-based culture systems cannot be
used to handle single batches of >10.sup.9 cells. A logical
further development from dishes are bag technologies, e.g. the Wave
Bioreactor, in which the medium volume is significantly enlarged by
using bags and cell attachment surface can be enlarged by using
buoyant carriers. However, bag-type reactors typically operate from
2.times.10.sup.6 to about 6.times.10.sup.6 cells/ml medium,
requiring significant media dilution during culture and a laborious
10-100 fold debulking. Moreover, bag technologies, and generally
all large-vessel stirred tank type bioreactors, do not provide
tissue-like physiologic environments that are conducive to "normal"
cell expansion and differentiation.
3. SUMMARY
[0005] Provided herein are methods of expanding hematopoietic cells
(e.g., hematopoietic stem cells or hematopoietic progenitor cells),
to differentiating the expanded hematopoietic cells into
administrable erythrocytes (red blood cells), and to the production
of administrable units of cells comprising the erythrocytes.
[0006] In one aspect, provided herein is a method of producing
erythrocytes. In one embodiment, the method comprises
differentiating hematopoietic cells from human placental perfusate
to erythrocytes. In a specific embodiment, the method comprises
expanding a population of hematopoietic cells in the absence of a
feeder layer, wherein the hematopoietic cells are obtained from
human placental perfusate and optionally from human umbilical cord
blood; subsequently expanding said hematopoietic cells in the
presence of a feeder layer; and differentiating the hematopoietic
cells to erythrocytes or progenitors of erythrocytes.
[0007] In another aspect, provided herein is a bioreactor for the
expansion of hematopoietic cells and differentiation of said
hematopoietic cells into erythrocytes. The bioreactor allows for
production of a number of erythrocytes equivalent to current
methods of producing erythrocytes, in a much smaller volume, by
facilitating a continuous erythrocyte production method rather than
a batch method. In specific embodiments, the bioreactor comprises a
cell culture element, a cell separation element, a gas provision
element and/or a medium provision element. In a specific embodiment
of the bioreactor, the erythrocytes are collected by magnetic bead
separation. In another embodiment of the method, the erythrocytes
are collected by partially or fully deoxygenating hemoglobin in
said erythrocytes, and attracting the erythrocytes to a surface
using a magnetic field.
[0008] In another aspect, provided herein is a method of the
production of erythrocytes using the bioreactor described herein.
In a specific embodiment, provided herein is a method of producing
erythrocytes comprising producing erythrocytes using a plurality of
the bioreactors disclosed herein. In other specific embodiments of
the method, the production of said erythrocytes is automated.
[0009] In one aspect, provided herein is a method of producing
erythrocytes, comprising (a) expanding a plurality of hematopoietic
cells in the absence of feeder cells, optionally in contact with an
immunomodulatory compound, wherein the immunomodulatory compound
increases the number of hematopoietic cells compared to a plurality
of hematopoietic cells not in contact with the immunomodulatory
compound, to produce a first expanded hematopoietic cell
population; (b) expanding the first expanded hematopoietic cell
population in the presence of a plurality of feeder cells to
produce a second expanded hematopoietic cell population; (c)
contacting said second expanded hematopoietic cell population with
one or more factors that cause differentiation of hematopoietic
cells in said second expanded hematopoietic cell population into
erythrocytes; and (d) isolating said erythrocytes from said second
expanded hematopoietic cell population. In a specific embodiment,
said isolating of erythrocytes in step (d) is performed
continuously. In other specific embodiments, said isolating of
erythrocytes in step (d) is performed periodically, e.g., every 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or
60 minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,
6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or more. In
another specific embodiment, said isolating of erythrocytes in step
(d) is performed periodically when one or more culture condition
criteria are met, e.g., achievement in the culture of a particular
cell density; achievement in the culture of a particular number of
cells per milliliter expressing certain erythrocyte markers, e.g.,
CD36 or glycophorin A; or the like.
[0010] In a specific embodiment, said hematopoietic cells are
CD34.sup.+. In another specific embodiment, said hematopoietic
cells are Thy-1.sup.+, CXCR4.sup.+, CD133.sup.+ or KDR.sup.+. In
another specific embodiment, said hematopoietic cells are
CD45.sup.-. In another specific embodiment, said hematopoietic
cells are HLA-DR.sup.-, CD71.sup.-, CD2.sup.-, CD3.sup.-,
CD11b.sup.-, CD11c.sup.-, CD14.sup.-, CD19.sup.-, CD16.sup.-,
CD24.sup.-, CD56.sup.-, CD66b.sup.- and/or glycophorin A.sup.-. In
another specific embodiment, said hematopoietic stem cells are
Lin.sup.-.
[0011] In another specific embodiment, said hematopoietic cells are
obtained from cord blood, placental blood, peripheral blood, or
bone marrow. In another specific embodiment, said hematopoietic
cells are obtained from placental perfusate. In another specific
embodiment, said hematopoietic cells are obtained from umbilical
cord blood and placental perfusate. In a more specific embodiment,
said placental perfusate is obtained by passage of perfusion
solution through only the vasculature of a placenta. In another
specific embodiment, said hematopoietic cells are human
hematopoietic cells.
[0012] In another specific embodiment, said feeder cells are from
the same individual as the hematopoietic cells. In another specific
embodiment, said feeder cells are from a different individual as
the hematopoietic cells. In a more specific embodiment, said feeder
cells are adherent placental stem cells, bone marrow-derived
mesenchymal stem cells, mesenchymal stem cells from peripheral
blood, mesenchymal stem cells from cord blood, or stromal stem
cells, or a combination of any of the foregoing. In another
specific embodiment, said feeder cells are adherent placental stem
cells. In a more specific embodiment, said adherent placental stem
cells are CD200.sup.+ or HLA-G.sup.+; CD73.sup.+, CD105.sup.+, and
CD200.sup.+; CD200.sup.+ and OCT-4.sup.+; CD73.sup.+, CD105.sup.+
and HLA-G.sup.+; CD73.sup.+ and CD105.sup.+ and facilitate the
formation of one or more embryoid-like bodies in a population of
isolated placental cells comprising said stem cells when said
population is cultured under conditions that allow formation of
embryoid-like bodies; or OCT-4.sup.+ and facilitate formation of
one or more embryoid-like bodies in a population of isolated
placental cells comprising said stem cell when cultured under
conditions that allow formation of embryoid-like bodies. In another
more specific embodiment, the adherent placental stem cells are
CD10.sup.+, CD34.sup.-, CD105.sup.+, and CD200.sup.+. In another
more specific embodiment, the adherent placental stem cells are
HLA-A,B,C.sup.+, CD45.sup.-, CD133.sup.- and CD34.sup.-. In another
more specific embodiment, the adherent placental stem cells are
CD10.sup.+, CD13.sup.+, CD33.sup.+, CD45.sup.-, CD117.sup.- and
CD133.sup.-. In another more specific embodiment, the adherent
placental stem cells are CD10.sup.-, CD33.sup.-, CD44.sup.+,
CD45.sup.-, and CD117.sup.-. In another more specific embodiment,
the adherent placental stem cells are HLA A,B,C.sup.+, CD45.sup.-,
CD34.sup.-, CD133.sup.-; positive for CD10, CD13, CD38, CD44, CD90,
CD105, CD200 and/or HLA-G, and/or negative for CD117. In another
more specific embodiment, the adherent placental stem cells are
CD200.sup.+ and CD10.sup.+, as determined by antibody binding, and
CD117.sup.-, as determined by both antibody binding and RT-PCR. In
another more specific embodiment, the adherent placental stem cells
are CD10.sup.+, CD29.sup.-, CD54.sup.+, CD200.sup.+, HLA-G.sup.+,
HLA class 1.sup.+ and .beta.-2-microglobulin.sup.+.
[0013] In certain embodiments of the method, a plurality of said
hematopoietic cells is blood type A, blood type O, blood type AB,
blood type O; is Rh positive or Rh negative; blood type M, blood
type N, blood type S, or blood type s; blood type P1; blood type
Lua, blood type Lub, or blood type Lu(a); blood type K (Kell), k
(cellano), Kpa, Kpb, K(a+), Kp(a-b-) or K-k-Kp(a-b-); blood type
Le(a-b-), Le(a+b-) or Le(a-b+); blood type Fy a, Fy b or Fy(a-b-);
or blood type Jk(a-b-), Jk(a+b-), Jk(a-b+) or Jk(a+b+). In a more
specific embodiment, the hematopoietic cells are type O, Rh
positive; type O, Rh negative; type A, Rh positive; type A, Rh
negative; type B, Rh positive; type B, Rh negative; type AB, Rh
positive or type AB, Rh negative. In other specific embodiments of
the method, greater than 90%, 95%, 98%, or 99%, or each, of said
hematopoietic cells is blood type A, blood type O, blood type AB,
blood type O; is Rh positive or Rh negative; blood type M, blood
type N, blood type S, or blood type s; blood type P1; blood type
Lua, blood type Lub, or blood type Lu(a); blood type K (Kell), k
(cellano), Kpa, Kpb, K(a+), Kp(a-b-) or K-k-Kp(a-b-); blood type
Le(a-b-), Le(a+b-) or Le(a-b+); blood type Fy a, Fy b or Fy(a-b-);
or blood type Jk(a-b-), Jk(a+b-), Jk(a-b+) or Jk(a+b+). In more
specific embodiments, the hematopoietic cells are type O, Rh+; type
O, Rh negative; type A, Rh positive; type A, Rh negative; type B,
Rh positive; type B, Rh negative; type AB, Rh positive or type AB,
Rh negative.
[0014] As used herein, the term "hematopoietic cells" includes
hematopoietic stem cells and hematopoietic progenitor cells, that
is, blood cells able to differentiate into erythrocytes.
[0015] As used herein, "+", when used to indicate the presence of a
particular cellular marker, means that the cellular marker is
detectably present in fluorescence activated cell sorting over an
isotype control; or is detectable above background in quantitative
or semi-quantitative RT-PCR.
[0016] As used herein, "-", when used to indicate the presence of a
particular cellular marker, means that the cellular marker is not
detectably present in fluorescence activated cell sorting over an
isotype control; or is not detectable above background in
quantitative or semi-quantitative RT-PCR.
4. BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1: Flow cytometric analysis of HPP-derived
CD34.sup.+/CD45.sup.- and CD34.sup.+/CD45.sup.+ cells.
[0018] FIGS. 2A-2B: Cell expansion in pomalidomide supplemented
IMDM medium. FIG. 2A: Fold expansion of total nucleated cells
(TNC). FIG. 2B: Fold expansion of CD34.sup.+ cells.
[0019] FIGS. 3A-3C: CFU-forming activity of expanded cultures. FIG.
3A: Fold expansion of TNC. FIG. 3B: Fold expansion of CD34.sup.+
cells. FIG. 3C: Expansion of CD34.sup.+ cells in number of cells.
"Compound 1" is pomalidomide.
[0020] FIGS. 4A-4B: Ex vivo expansion of CD34.sup.+ cells from HPP
(FIG. 4A) and umbilical cord blood (UCB) (FIG. 4B) in
cytokine-supplemented RPMI Medium (see Example 3).
[0021] FIGS. 5A-5D: Giemsa staining of CD34.sup.+ cultures in
cytokine supplemented RPMI medium. FIG. 5A: CD34.sup.+ cells at day
0, showing medium blue color. FIG. 5B: Basophilic normoblasts at
day 9, showing medium blue color. FIG. 5C: Orthochromatophilic
normoblasts at day 11, showing medium blue color. FIG. 5D:
Polychromatophilic erythrocytes/erythrocytes at day 21, showing
pink color. All images were obtained at magnification
400.times..
5. DETAILED DESCRIPTION
[0022] Provided herein is a method of producing erythrocytes from
expanded hematopoietic cells, e.g., hematopoietic stem cells and/or
hematopoietic progenitor cells. In one embodiment, hematopoietic
cells are collected from a source of such cells, e.g., placental
perfusate and umbilical cord blood. The hematopoietic cells are
expanded first without the use of feeder cells. Such isolation and
expansion can be performed in a central facility, which provides
expanded hematopoietic cells for shipment to decentralized
expansion and differentiation at points of use, e.g., hospital,
military base, military front line, or the like. Expansion at
point-of-use, in a preferred embodiment, is accomplished using
feeder cells. Feeder cell-dependent expansion, according to the
methods in Section 5.2.2, below, preferably takes place within a
bioreactor, as exemplified herein. Differentiation of erythrocytes,
according to the methods of Section 5.3, below, also preferably
takes place in the bioreactor. Collection of erythrocytes produced
in the method, in a preferred embodiment, is performed continuously
or periodically, e.g., during differentiation. The continuous or
periodic separation aspect of the method allows for the production
of erythrocytes in a substantially smaller space than possible
using, e.g., batch methods. The time for collection and expansion
of the hematopoietic cells is approximately 5-10 days, typically
about 7 days. Erythrocytes are purified on-site and packaged into
administrable units.
[0023] In one aspect, provided herein is a method of producing
erythrocytes, comprising (a) expanding a plurality of hematopoietic
cells in the absence of feeder cells, optionally in contact with an
immunomodulatory compound, wherein the immunomodulatory compound
increases the number of hematopoietic cells compared to a plurality
of hematopoietic cells not in contact with the immunomodulatory
compound, to produce a first expanded hematopoietic cell
population; (b) expanding the first expanded hematopoietic cell
population in the presence of a plurality of feeder cells to
produce a second expanded hematopoietic cell population; (c)
contacting said second expanded hematopoietic cell population with
one or more factors that cause differentiation of hematopoietic
cells in said second expanded hematopoietic cell population into
erythrocytes; and (d) isolating said erythrocytes from said second
expanded hematopoietic cell population. In a specific embodiment,
said isolating of erythrocytes in step (d) is performed
continuously. In other specific embodiments, said isolating of
erythrocytes in step (d) is performed periodically, e.g., every 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or
60 minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,
6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or more. In
another specific embodiment, said isolating of erythrocytes in step
(d) is performed periodically when one or more culture condition
criteria are met, e.g., achievement in the culture of a particular
cell density; achievement in the culture of a particular number of
cells per milliliter expressing certain erythrocyte markers, e.g.,
CD36 or glycophorin A; or the like.
[0024] 5.1. Hematopoietic Cells
[0025] Hematopoietic cells useful in the methods disclosed herein
can be any hematopoietic cells able to differentiate into
erythrocytes, e.g., precursor cells, hematopoietic progenitor
cells, hematopoietic stem cells, or the like. Hematopoietic cells
can be obtained from tissue sources such as, e.g., bone marrow,
cord blood, placental blood, peripheral blood, or the like.
Hematopoietic cells can be obtained from placenta. In a specific
embodiment, the hematopoietic cells are obtained from placental
perfusate. Hematopoietic cells from placental perfusate can
comprise a mixture of fetal and maternal hematopoietic cells, e.g.,
a mixture in which maternal cells comprise greater than 5% of the
total number of hematopoietic cells. Preferably, hematopoietic
cells from placental perfusate comprise at least about 90%, 95%,
98%, 99% or 99.5% fetal cells.
[0026] In certain embodiments, the hematopoietic cells are
CD34.sup.+ cells. CD34.sup.+ hematopoietic cells can, in certain
embodiments, express or lack the cellular marker CD38. Thus, in
specific embodiments, the hematopoietic cells useful in the methods
disclosed herein are CD34.sup.+CD38.sup.+ or CD34.sup.+CD38.sup.-.
In a more specific embodiment, the hematopoietic cell is
CD34.sup.+CD38.sup.-Lin.sup.-. In another specific embodiment, the
hematopoietic cell is one or more of CD2.sup.-, CD3.sup.-,
CD11b.sup.-, CD11c.sup.-, CD14.sup.-, CD16.sup.-, CD19.sup.-,
CD24.sup.-, CD56.sup.-, CD66b.sup.- and glycophorin A.sup.-. In
another specific embodiment, the hematopoietic cell is CD2.sup.-,
CD3.sup.-, CD11b.sup.-, CD11c.sup.-, CD14.sup.-, CD16.sup.-,
CD19.sup.-, CD24.sup.-, CD56.sup.-, CD66b.sup.- and glycophorin
A.sup.-. In another more specific embodiment, the hematopoietic
cell is CD34.sup.+CD38.sup.-CD33.sup.-CD117.sup.-. In another more
specific embodiment, the hematopoietic cell is
CD34.sup.+CD38.sup.-CD33.sup.-CD117.sup.-CD235.sup.-CD36.sup.-.
[0027] In another embodiment, the hematopoietic cells are
CD45.sup.-. In a specific embodiment, the hematopoietic cells are
CD34.sup.+CD45.sup.-. In another specific embodiment, the
hematopoietic cells are CD34.sup.+CD45.sup.+.
[0028] In another embodiment, the hematopoietic cell is
Thy-1.sup.+. In a specific embodiment, the hematopoietic cell is
CD34.sup.+Thy-1.sup.+. In another embodiment, the hematopoietic
cells are CD133.sup.+. In specific embodiments, the hematopoietic
cells are CD34.sup.+CD133.sup.+ or CD133.sup.+Thy-1.sup.+. In
another specific embodiment, the CD34.sup.+ hematopoietic cells are
CXCR4.sup.+. In another specific embodiment, the CD34.sup.+
hematopoietic cells are CXCR4.sup.-. In another embodiment, the
hematopoietic cells are positive for KDR (vascular growth factor
receptor 2). In specific embodiments, the hematopoietic cells are
CD34.sup.+KDR.sup.+, CD133.sup.+KDR.sup.+ or Thy-1.sup.+KDR.sup.+.
In certain other embodiments, the hematopoietic cells are positive
for aldehyde dehydrogenase (ALDH.sup.+), e.g., the cells are
CD34.sup.+ALDH.sup.+.
[0029] In certain embodiments, the hematopoietic cells are
CD34.sup.-.
[0030] The hematopoietic cells can also lack certain markers that
indicate lineage commitment, or a lack of developmental naivete.
For example, in another embodiment, the hematopoietic cells are
HLA-DR.sup.-. In specific embodiments, the hematopoietic cells are
CD34.sup.+HLA-DR.sup.-, CD133.sup.+HLA-DR.sup.-,
Thy-1.sup.+HLA-DR.sup.- or ALDH.sup.+HLA-DR.sup.- In another
embodiment, the hematopoietic cells are negative for one or more,
preferably all, of lineage markers CD2, CD3, CD11b, CD11c, CD14,
CD16, CD19, CD24, CD56, CD66b and glycophorin A.
[0031] Thus, populations of hematopoietic cells can be selected for
use in the methods disclosed herein on the basis of the presence of
markers that indicate an undifferentiated state, or on the basis of
the absence of lineage markers indicating that at least some
lineage differentiation has taken place. Methods of isolating cells
on the basis of the presence or absence of specific markers is
discussed in detail, e.g., in Section 5.1.2, below.
[0032] Hematopoietic cells used in the methods provided herein can
be a substantially homogeneous population, e.g., a population
comprising at least about 95%, at least about 98% or at least about
99% hematopoietic cells from a single tissue source, or a
population comprising hematopoietic cells exhibiting the same
hematopoietic cell-associated cellular markers. For example, in
various embodiment, the hematopoietic cells can comprise at least
about 95%, 98% or 99% hematopoietic cells from bone marrow, cord
blood, placental blood, peripheral blood, or placenta, e.g.,
placenta perfusate.
[0033] Hematopoietic cells used in the methods provided herein can
be obtained from a single individual, e.g., from a single placenta,
or from a plurality of individuals, e.g., can be pooled. Where the
hematopoietic cells are obtained from a plurality of individuals
and pooled, it is preferred that the hematopoietic cells be
obtained from the same tissue source. Thus, in one embodiment, the
pooled hematopoietic cells are all from placenta, e.g., placental
perfusate, all from placental blood, all from umbilical cord blood,
all from peripheral blood, and the like.
[0034] Hematopoietic cells used in the methods disclosed herein can
comprise hematopoietic cells from two or more tissue sources.
Preferably, when hematopoietic cells from two or more sources are
combined for use in the methods herein, a plurality of the
hematopoietic cells used to produce erythrocytes comprise
hematopoietic cells from placenta, e.g., placenta perfusate. In
various embodiments, the hematopoietic cells used to produce
erythrocytes comprise hematopoietic cells from placenta and from
cord blood; from placenta and peripheral blood; form placenta and
placental blood, or placenta and bone marrow. In a preferred
embodiment, the hematopoietic cells comprise hematopoietic cells
from placental perfusate in combination with hematopoietic cells
from cord blood, wherein the cord blood and placenta are from the
same individual, i.e., wherein the perfusate and cord blood are
matched. In embodiments in which the hematopoietic cells comprise
hematopoietic cells from two tissue sources, the hematopoietic
cells from the sources can be combined in a ratio of, for example,
1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3, 9:2, 1:10, 1:9, 1:8, 1:7,
1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or
9:1.
[0035] Preferably, the erythrocytes produced from hematopoietic
cells according to the methods provided herein are homogeneous with
respect to blood type, e.g., identical with respect to cell surface
markers, antigens, or the like. Such homogeneity can be achieved,
for example, by obtaining hematopoietic cells from a single
individual of the desired blood type. In embodiments in which
hematopoietic cells are pooled from a plurality of individuals, it
is preferred that each of the individuals shares at least one, at
least two, or at least three or more antigenic blood determinants
in common. In various embodiments, for example, the individual from
which the hematopoietic cells are obtained is, or each of the
individuals from which hematopoietic cells are obtained are, blood
type O, blood type A, blood type B, or blood type AB. In other
embodiments, the individual from which the hematopoietic cells are
obtained is, or each of the individuals from which hematopoietic
cells are obtained are, Rh positive, or Rh negative. In a specific
embodiment, the individual from which the hematopoietic cells are
obtained is, or each of the individuals from which hematopoietic
cells are obtained are, O positive and Rh negative. In more
specific embodiments, the individual from which the hematopoietic
cells are obtained is, or each of the individuals from which
hematopoietic cells are obtained are, O positive, O negative, A
positive, A negative, B positive, B negative, AB positive, or AB
negative. In other specific embodiments, the individual from which
the hematopoietic cells are obtained is, or each of the individuals
from which hematopoietic cells are obtained are, blood type M,
blood type N, blood type S, or blood type s. In other specific
embodiments, the individual from which the hematopoietic cells are
obtained is, or each of the individuals from which hematopoietic
cells are obtained are, blood type P1. In other specific
embodiments, the individual from which the hematopoietic cells are
obtained is, or each of the individuals from which hematopoietic
cells are obtained are, blood type Lua, blood type Lub, or blood
type Lu(a). In other specific embodiments, the individual from
which the hematopoietic cells are obtained is, or each of the
individuals from which hematopoietic cells are obtained are, blood
type K (Kell), k (cellano), Kpa, Kpb, K(a+), Kp(a-b-) or
K-k-Kp(a-b-). In other specific embodiments, the individual from
which the hematopoietic cells are obtained is, or each of the
individuals from which hematopoietic cells are obtained are, blood
type Le(a-b-), Le(a+b-) or Le(a-b+). In other specific embodiments,
the individual from which the hematopoietic cells are obtained is,
or each of the individuals from which hematopoietic cells are
obtained are, blood type Fy a, Fy b or Fy(a-b-). In other specific
embodiments, the individual from which the hematopoietic cells are
obtained is, or each of the individuals from which hematopoietic
cells are obtained are, blood type Jk(a-b-), Jk(a+b-), Jk(a-b+) or
Jk(a+b+). In other specific embodiments, the individual from whom
the hematopoietic cells are obtained is classifiable within blood
group Diego, Cartwright, Xgm Scianna, Bombrock, Colton,
Lansteiner-Weiner, Chido/Rogers, Hh, Kx, Gergich, Cromer, Knops,
Indian, Ok, Raph, or JMH. In other specific embodiments, each of
the individuals from which hematopoietic cells are obtained are of
the same blood type within a blood typing system or group of
antigenic determinants, wherein said blood typing system or group
of antigenic determinants are Diego, Cartwright, Xgm Scianna,
Bombrock, Colton, Lansteiner-Weiner, Chido/Rogers, Hh, Kx, Gergich,
Cromer, Knops, Indian, Ok, Raph, or JMH.
[0036] 5.1.1. Placental Hematopoietic Stem Cells
[0037] In certain embodiment, the hematopoietic cells used in the
methods provided herein are placental hematopoietic cells. As used
herein, "placental hematopoietic cells" means hematopoietic cells
obtained from the placenta itself, and not from placental blood or
from umbilical cord blood. In one embodiment, placental
hematopoietic cells are CD34.sup.+. In a specific embodiment, the
placental hematopoietic cells are predominantly (e.g., at least
about 90%, 95% or 98%) CD34.sup.+CD38.sup.- cells. In another
specific embodiment, the placental hematopoietic cells are
predominantly (e.g., at least about 90%, 95% or 98%)
CD34.sup.+CD38.sup.+ cells. Placental hematopoietic cells can be
obtained from a post-partum mammalian (e.g., human) placenta by any
means known to those of skill in the art, e.g., by perfusion.
[0038] In another embodiment, the placental hematopoietic cell is
CD45.sup.-. In a specific embodiment, the hematopoietic cell is
CD34.sup.+CD45.sup.-. In another specific embodiment, the placental
hematopoietic cells are CD34.sup.+CD45.sup.+.
[0039] 5.1.1.1. Obtaining Placental Hematopoietic Cells by
Perfusion
[0040] Placental hematopoietic cells can be obtained using
perfusion. Methods of perfusing mammalian placenta to obtain cells,
including placental hematopoietic cells, are disclosed, e.g., in
U.S. Pat. No. 7,045,148, entitled "Method of Collecting placental
Stem Cells," U.S. Pat. No. 7,255,879, entitled "Post-Partum
Mammalian Placenta, Its Use and Placental Stem Cells Therefrom,"
and in U.S. Application No. 2007/0190042, entitled "Improved Medium
for Collecting Placental Stem Cells and Preserving Organs," the
disclosures of which are hereby incorporated by reference in their
entireties.
[0041] Placental hematopoietic cells can be collected by perfusion,
e.g., through the placental vasculature, using, e.g., a saline
solution (for example, phosphate-buffered saline, a 0.9% NaCl
solution, or the like), culture medium or organ preservation
solution as a perfusion solution. In one embodiment, a mammalian
placenta is perfused by passage of perfusion solution through
either or both of the umbilical artery and umbilical vein. The flow
of perfusion solution through the placenta may be accomplished
using, e.g., gravity flow into the placenta. Preferably, the
perfusion solution is forced through the placenta using a pump,
e.g., a peristaltic pump. The umbilical vein can be, e.g.,
cannulated with a cannula, e.g., a TEFLON.RTM. or plastic cannula,
that is connected to a sterile connection apparatus, such as
sterile tubing, which, in turn is connected to a perfusion
manifold.
[0042] In preparation for perfusion, the placenta is preferably
oriented (e.g., suspended) in such a manner that the umbilical
artery and umbilical vein are located at the highest point of the
placenta. The placenta can be perfused by passage of a perfusion
fluid through the placental vasculature and surrounding tissue. The
placenta can also be perfused by passage of a perfusion fluid into
the umbilical vein and collection from the umbilical arteries, or
passage of a perfusion fluid into the umbilical arteries and
collection from the umbilical vein.
[0043] In one embodiment, for example, the umbilical artery and the
umbilical vein are connected simultaneously, e.g., to a pipette
that is connected via a flexible connector to a reservoir of the
perfusion solution. The perfusion solution is passed into the
umbilical vein and artery. The perfusion solution exudes from
and/or passes through the walls of the blood vessels into the
surrounding tissues of the placenta, and is collected in a suitable
open vessel, e.g., a sterile pan, from the surface of the placenta
that was attached to the uterus of the mother during gestation. The
perfusion solution may also be introduced through the umbilical
cord opening and allowed to flow or percolate out of openings in
the wall of the placenta which interfaced with the maternal uterine
wall. Placental cells that are collected by this method, which can
be referred to as a "pan" method, are typically a mixture of fetal
and maternal cells.
[0044] In another embodiment, the perfusion solution is passed
through the umbilical veins and collected from the umbilical
artery, or is passed through the umbilical artery and collected
from the umbilical veins. Placental cells collected by this method,
which can be referred to as a "closed circuit" method, are
typically almost exclusively fetal.
[0045] The closed circuit perfusion method can, in one embodiment,
be performed as follows. A post-partum placenta is obtained within
about 48 hours after birth. The umbilical cord is clamped and cut
above the clamp. The umbilical cord can be discarded, or can
processed to recover, e.g., umbilical cord stem cells, and/or to
process the umbilical cord membrane for the production of a
biomaterial. The amniotic membrane can be retained during
perfusion, or can be separated from the chorion, e.g., using blunt
dissection with the fingers. If the amniotic membrane is separated
from the chorion prior to perfusion, it can be, e.g., discarded, or
processed, e.g., to obtain stem cells by enzymatic digestion, or to
produce, e.g., an amniotic membrane biomaterial, e.g., the
biomaterial described in U.S. Application Publication No.
2004/0048796.
[0046] After cleaning the placenta of all visible blood clots and
residual blood, e.g., using sterile gauze, the umbilical cord
vessels are exposed, e.g., by partially cutting the umbilical cord
membrane to expose a cross-section of the cord. The vessels are
identified, and opened, e.g., by advancing a closed alligator clamp
through the cut end of each vessel. The apparatus, e.g., plastic
tubing connected to a perfusion device or peristaltic pump, is then
inserted into each of the placental arteries. The pump can be any
pump suitable for the purpose, e.g., a peristaltic pump. Plastic
tubing, connected to a sterile collection reservoir, e.g., a blood
bag such as a 250 mL collection bag, is then inserted into the
placental vein. Alternatively, the tubing connected to the pump is
inserted into the placental vein, and tubes to a collection
reservoir(s) are inserted into one or both of the placental
arteries. The placenta is then perfused with a volume of perfusion
solution, e.g., about 750 ml of perfusion solution. Cells in the
perfusate are then collected, e.g., by centrifugation.
[0047] In one embodiment, the proximal umbilical cord is clamped
during perfusion, and more preferably, is clamped within 4-5 cm
(centimeter) of the cord's insertion into the placental disc.
[0048] The first collection of perfusion fluid from a mammalian
placenta during the exsanguination process is generally colored
with residual red blood cells of the cord blood and/or placental
blood. The perfusion fluid becomes more colorless as perfusion
proceeds and the residual cord blood cells are washed out of the
placenta. Generally from 30 to 100 ml (milliliter) of perfusion
fluid is adequate to initially exsanguinate the placenta, but more
or less perfusion fluid may be used depending on the observed
results.
[0049] The volume of perfusion liquid used to collect placental
hematopoietic cells may vary depending upon the number of
hematopoietic cells to be collected, the size of the placenta, the
number of collections to be made from a single placenta, etc. In
various embodiments, the volume of perfusion liquid may be from 50
mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000
mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL.
Typically, the placenta is perfused with 700-800 mL of perfusion
liquid following exsanguination.
[0050] The placenta can be perfused a plurality of times over the
course of several hours or several days to obtain placental
hematopoietic cells. Where the placenta is to be perfused a
plurality of times, it may be maintained or cultured under aseptic
conditions in a container or other suitable vessel, and perfused
with a stem cell collection composition (see U.S. Application
Publication No. 2007/0190042, the disclosure of which is
incorporated herein by reference in its entirety), or a standard
perfusion solution (e.g., a normal saline solution such as
phosphate buffered saline ("PBS")) with or without an anticoagulant
(e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin),
and/or with or without an antimicrobial agent (e.g.,
.beta.-mercaptoethanol (0.1 mM); antibiotics such as streptomycin
(e.g., at 40-100 .mu.g/ml), penicillin (e.g., at 40 U/ml),
amphotericin B (e.g., at 0.5 .mu.g/ml). In one embodiment, an
isolated placenta is maintained or cultured for a period of time
without collecting the perfusate, such that the placenta is
maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3
or more days before perfusion and collection of perfusate. The
perfused placenta can be maintained for one or more additional
time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused a
second time with, e.g., 700-800 mL perfusion fluid. The placenta
can be perfused 1, 2, 3, 4, 5 or more times, for example, once
every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment,
perfusion of the placenta and collection of perfusion solution,
e.g., stem cell collection composition, is repeated until the
number of recovered nucleated cells falls below 100 cells/ml. The
perfusates at different time points can be further processed
individually to recover time-dependent populations of cells, e.g.,
placental hematopoietic cells. Perfusates from different time
points can also be pooled.
[0051] 5.1.1.2. Obtaining Placental Hematopoietic Cells by Tissue
Disruption
[0052] Hematopoietic cells can be isolated from placenta by
perfusion with a solution comprising one or more proteases or other
tissue-disruptive enzymes (e.g., trypsin, collagenase, papain,
chymotrypsin, subtilisin, hyaluronidase; a cathepsin, a caspase, a
calpain, chymosin, plasmepsin, pepsin, or the like). In a specific
embodiment, a placenta or portion thereof (e.g., amniotic membrane,
amnion and chorion, placental lobule or cotyledon, umbilical cord,
or combination of any of the foregoing) is brought to 25-37.degree.
C., and is incubated with one or more tissue-disruptive enzymes in
200 mL of a culture medium for 30 minutes. Cells from the perfusate
are collected, brought to 4.degree. C., and washed with a cold
inhibitor mix comprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM
beta-mercaptoethanol. The stem cells are washed after several
minutes with cold (e.g., 4.degree. C.) stem cell collection
composition.
[0053] In one embodiment, the placenta can be disrupted
mechanically (e.g., by crushing, blending, dicing, mincing or the
like) to obtain the hematopoietic cells. The placenta can be used
whole, or can be dissected into components prior to physical
disruption and/or enzymatic digestion and hematopoietic cell
recovery. For example, hematopoietic cells can be obtained from the
amniotic membrane, chorion, umbilical cord, placental cotyledons,
or any combination thereof.
[0054] Placental hematopoietic cells can also be obtained by
enzymatic disruption of the placenta using a tissue-disrupting
enzyme, e.g., trypsin, collagenase, papain, chymotrypsin,
subtilisin, hyaluronidase; a cathepsin, a caspase, a calpain,
chymosin, plasmepsin, pepsin, or the like. Enzymatic digestion
preferably uses a combination of enzymes, e.g., a combination of a
matrix metalloprotease and a neutral protease, for example, a
combination of collagenase and dispase. In one embodiment,
enzymatic digestion of placental tissue uses a combination of a
matrix metalloprotease, a neutral protease, and a mucolytic enzyme
for digestion of hyaluronic acid, such as a combination of
collagenase, dispase, and hyaluronidase or a combination of
LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) and
hyaluronidase. Other enzymes that can be used to disrupt placenta
tissue include papain, deoxyribonucleases, serine proteases, such
as trypsin, chymotrypsin, or elastase. Serine proteases may be
inhibited by alpha 2 microglobulin in serum and therefore the
medium used for digestion is usually serum-free. EDTA and DNase are
commonly used in enzyme digestion procedures to increase the
efficiency of cell recovery. The digestate is preferably diluted so
as to avoid trapping stem cells within the viscous digest.
[0055] Any combination of tissue digestion enzymes can be used.
Typical concentrations for tissue digestion enzymes include, e.g.,
50-200 U/mL for collagenase I and collagenase IV, 1-10 U/mL for
dispase, and 10-100 U/mL for elastase. Proteases can be used in
combination, that is, two or more proteases in the same digestion
reaction, or can be used sequentially in order to liberate
placental stem cells. For example, in one embodiment, a placenta,
or part thereof, is digested first with an appropriate amount of
collagenase I at 2 mg/ml for 30 minutes, followed by digestion with
trypsin, 0.25%, for 10 minutes, at 37.degree. C. Serine proteases
are preferably used consecutively following use of other
enzymes.
[0056] In another embodiment, the tissue can further be disrupted
by the addition of a chelator, e.g., ethylene glycol
bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) or
ethylenediaminetetraacetic acid (EDTA) to the stem cell collection
composition comprising the stem cells, or to a solution in which
the tissue is disrupted and/or digested prior to isolation of the
placental hematopoietic cells.
[0057] It will be appreciated that where an entire placenta, or
portion of a placenta comprising both fetal and maternal cells (for
example, where the portion of the placenta comprises the chorion or
cotyledons), the placental hematopoietic cells collected will
comprise a mix of placental stem cells derived from both fetal and
maternal sources. Where a portion of the placenta that comprises
no, or a negligible number of, maternal cells (for example,
amnion), the placental stem cells collected will comprise almost
exclusively fetal placental stem cells.
[0058] 5.1.2. Isolation, Sorting, and Characterization of Cells
[0059] Cells, including hematopoietic cells from any source, e.g.,
mammalian placenta, can initially be purified from (i.e., be
isolated from) other cells by Ficoll gradient centrifugation. Such
centrifugation can follow any standard protocol for centrifugation
speed, etc. In one embodiment, for example, cells collected from
the placenta are recovered from perfusate by centrifugation at
5000.times. g for 15 minutes at room temperature, which separates
cells from, e.g., contaminating debris and platelets. In another
embodiment, placental perfusate is concentrated to about 200 ml,
gently layered over Ficoll, and centrifuged at about 1100.times. g
for 20 minutes at 22.degree. C., and the low-density interface
layer of cells is collected for further processing.
[0060] Cell pellets can be resuspended in, e.g., fresh saline
solution, or a medium suitable for stem cell maintenance, e.g.,
IMDM serum-free medium containing 2 U/ml heparin and 2 mM EDTA
(GibcoBRL, NY). The total mononuclear cell fraction can be
isolated, e.g., using LYMPHOPREP.RTM. (Nycomed Pharma, Oslo,
Norway) according to the manufacturer's recommended procedure.
[0061] As used herein, "isolating" cells, including placental
cells, e.g., placental hematopoietic cells or placental stem cells,
means to remove at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95% or 99% of the cells with which the isolated cells are
normally associated in the intact tissue, e.g., mammalian placenta.
A cell from an organ is "isolated" when the cell is present in a
population of cells that comprises fewer than 50% of the cells with
which the stem cell is normally associated in the intact organ.
[0062] Adherent placental cells obtained by perfusion or digestion,
e.g., for use as feeder cells, can, for example, be further, or
initially, isolated by differential trypsinization using, e.g., a
solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.).
Differential trypsinization is possible because placental stem
cells typically detach from plastic surfaces within about five
minutes whereas other adherent populations typically require more
than 20-30 minutes incubation. The detached placental stem cells
can be harvested following trypsinization and trypsin
neutralization, using, e.g., Trypsin Neutralizing Solution (TNS,
Cambrex). In one embodiment of isolation of adherent cells,
aliquots of, for example, about 5-10.times.10.sup.6 cells are
placed in each of several T-75 flasks, preferably
fibronectin-coated T75 flasks. In such an embodiment, the cells can
be cultured with commercially available Mesenchymal Stem Cell
Growth Medium (MSCGM) (Cambrex), and placed in a tissue culture
incubator (37.degree. C., 5% CO.sub.2). After 10 to 15 days,
non-adherent cells are removed from the flasks by washing with PBS.
The PBS is then replaced by MSCGM. Flasks are preferably examined
daily for the presence of various adherent cell types and in
particular, for identification and expansion of clusters of
fibroblastoid cells.
[0063] The number and type of cells collected from a mammalian
placenta can be monitored, for example, by measuring changes in
morphology and cell surface markers using standard cell detection
techniques such as flow cytometry, cell sorting,
immunocytochemistry (e.g., staining with tissue specific or
cell-marker specific antibodies) fluorescence activated cell
sorting (FACS), magnetic activated cell sorting (MACS), by
examination of the morphology of cells using light or confocal
microscopy, and/or by measuring changes in gene expression using
techniques well known in the art, such as PCR and gene expression
profiling. These techniques can be used, too, to identify cells
that are positive for one or more particular markers. For example,
using antibodies to CD34, one can determine, using the techniques
above, whether a cell comprises a detectable amount of CD34, in an
assay such as an ELISA or RIA, or by FACS; if so, the cell is
CD34.sup.+. Likewise, if a cell, e.g., a feeder cell, e.g., an
adherent placental stem cell, produces enough OCT-4 RNA to be
detectable by RT-PCR, or significantly more OCT-4 RNA than an adult
cell, the cell is OCT-4.sup.+. Antibodies to cell surface markers
(e.g., CD markers such as CD34) and the sequence of stem
cell-specific genes, such as OCT-4, are well-known in the art.
[0064] Placental cells, particularly cells that have been isolated
by Ficoll separation, differential adherence, or a combination of
both, may be sorted using fluorescence activated cell sorting
(FACS). FACS is a well-known method for separating particles,
including cells, based on the fluorescent properties of the
particles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laser
excitation of fluorescent moieties in the individual particles
results in a small electrical charge allowing electromagnetic
separation of positive and negative particles from a mixture. In
one embodiment, cell surface marker-specific antibodies or ligands
are labeled with distinct fluorescent labels. Cells are processed
through the cell sorter, allowing separation of cells based on
their ability to bind to the antibodies used. FACS sorted particles
may be directly deposited into individual wells of 96-well or
384-well plates to facilitate separation and cloning.
[0065] In one embodiment, stem cells from placenta are sorted,
e.g., isolated, on the basis of expression one or more of the
markers CD34, CD38, CD44, CD45, CD73, CD105, CD117, CD200, OCT-4
and/or HLA-G.
[0066] In another embodiment, hematopoietic cells, e.g.,
CD34.sup.+, CD133.sup.+, KDR.sup.+ or Thy-1.sup.+ cells, are
sorted, e.g., isolated, on the basis of markers characteristic of
undifferentiated hematopoietic cells. Such sorting can be done,
e.g., in a population of cells that has not been sorted, e.g., a
population of cells from a perfusion or a tissue digestion, wherein
CD34.sup.+ cells represent a minority of the cells present in the
population. Such sorting can also be done in a population of cells
that is mostly (e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%,
98% or 99%) hematopoietic cells as, for example, a purification
step. For example, in a specific embodiment, CD34.sup.+ cells,
KDR.sup.+ cells, Thy-1.sup.+ cells, and/or CD133.sup.+ cells are
retained during sorting to produce a population of undifferentiated
hematopoietic cells.
[0067] In another embodiment, cells, e.g., hematopoietic cells are
sorted, e.g., excluded, on the basis of markers of
lineage-differentiated cells. For example, cells, in a population
of hematopoietic cells, that are CD2.sup.+, CD3.sup.+, CD11b.sup.+,
CD11c.sup.+, CD14.sup.+, CD16.sup.+, CD19.sup.+, CD24.sup.+,
CD56.sup.+, CD66b.sup.+ and/or glycophorin A.sup.+ are excluded
during sorting from the population of hematopoietic cells to
produce a population of undifferentiated hematopoietic cells.
[0068] In another embodiment, hematopoietic cells can be sorted,
e.g., isolated, on the basis of lack of expression of, e.g.,
lineage markers. In a specific embodiment, for example,
hematopoietic cells, e.g., CD34.sup.+ cells, can be isolated based
on a determination that the cells are one or more of CD2.sup.-,
CD3.sup.-, CD11b.sup.-, CD11c.sup.-, CD14.sup.-, CD16.sup.-,
CD19.sup.-, CD24.sup.-, CD56.sup.-, CD66b.sup.- and/or glycophorin
A.sup.-.
[0069] In another embodiment, cells, e.g., adherent placental stem
cells, are sorted first on the basis of their expression of CD34,
wherein CD34.sup.- cells are retained. In a specific embodiment,
CD34.sup.- cells that are CD200.sup.+HLA-G.sup.+ are separated from
all other CD34.sup.- cells. In another embodiment, cells from
placenta are based on their expression of markers CD200 and/or
HLA-G; for example, cells displaying either of these markers are
isolated for further use. Cells that express, e.g., CD200 and/or
HLA-G can, in a specific embodiment, be further sorted based on
their expression of CD73 and/or CD105, or epitopes recognized by
antibodies SH2, SH3 or SH4, or lack of expression of CD34, CD38 or
CD45. For example, in one embodiment, placental cells are sorted by
expression, or lack thereof, of CD200, HLA-G, CD73, CD105, CD34,
CD38 and CD45, and placental cells that are CD200.sup.+,
HLA-G.sup.+, CD73.sup.+, CD105.sup.+, CD34.sup.-, CD38.sup.- and
CD45.sup.- are isolated from other placental cells for further
use.
[0070] In another embodiment, magnetic beads can be used to
separate cells, e.g., DYNABEADS.RTM. (Invitrogen). The cells may be
sorted using a magnetic activated cell sorting (MACS) technique, a
method for separating particles based on their ability to bind
magnetic beads (0.5-100 .mu.m diameter). A variety of useful
modifications can be performed on the magnetic microspheres,
including covalent addition of antibody that specifically
recognizes a particular cell surface molecule or hapten. The beads
are then mixed with the cells to allow binding. Cells are then
passed through a magnetic field to separate out cells having the
specific cell surface marker. In one embodiment, these cells can
then isolated and re-mixed with magnetic beads coupled to an
antibody against additional cell surface markers. The cells are
again passed through a magnetic field, isolating cells that bound
both the antibodies. Such cells can then be diluted into separate
dishes, such as microtiter dishes for clonal isolation.
[0071] Adherent placental stem cells, e.g., to be used as feeder
cells, can also be characterized and/or sorted based on cell
morphology and growth characteristics. For example, placental stem
cells can be characterized as having, and/or selected on the basis
of, e.g., a fibroblastoid appearance in culture. Placental stem
cells can also be characterized as having, and/or be selected, on
the basis of their ability to form embryoid-like bodies. In one
embodiment, for example, placental cells that are fibroblastoid in
shape, express CD73 and CD105, and produce one or more
embryoid-like bodies in culture are isolated from other placental
cells. In another embodiment, OCT-4.sup.+ placental cells that
produce one or more embryoid-like bodies in culture are isolated
from other placental cells.
[0072] In another embodiment, placental stem cells, e.g., placental
hematopoietic cells or adherent placental stem cells, can be
identified and characterized by a colony forming unit assay. Colony
forming unit assays are commonly known in the art, such as MESEN
CULT.TM. medium (Stem Cell Technologies, Inc., Vancouver British
Columbia)
[0073] Placental stem cells can be assessed for viability,
proliferation potential, and longevity using standard techniques
known in the art, such as trypan blue exclusion assay, fluorescein
diacetate uptake assay, propidium iodide uptake assay (to assess
viability); and thymidine uptake assay, MTT cell proliferation
assay (to assess proliferation). Longevity may be determined by
methods well known in the art, such as by determining the maximum
number of population doubling in an extended culture.
[0074] Placental stem cells can also be separated from other
placental cells using other techniques known in the art, e.g.,
selective growth of desired cells (positive selection), selective
destruction of unwanted cells (negative selection); separation
based upon differential cell agglutinability in the mixed
population as, for example, with soybean agglutinin; freeze-thaw
procedures; filtration; conventional and zonal centrifugation;
centrifugal elutriation (counter-streaming centrifugation); unit
gravity separation; countercurrent distribution; electrophoresis;
and the like.
[0075] 5.2. Expansion of Hematopoietic Cells
[0076] Once a population of hematopoietic cells is obtained, the
population is expanded. One unit of erythrocytes is expected to
comprise about 1-2.times.10.sup.12 red blood cells. Hematopoietic
stem cell population doubling requires approximately 36 hours.
Thus, starting from about 5.times.10.sup.7 hematopoietic cells
according to standard methods, and assuming 100% efficiency in
expansion and differentiation, production of a unit of erythrocytes
would require approximately 14 hematopoietic cell population
doublings, or approximately 3 weeks. The method described in detail
below improves on standard methods by improving the culture
conditions of hematopoietic cells and increasing the number of
hematopoietic cells during expansion per unit time.
[0077] 5.2.1. Shortened Hematopoietic Cell Expansion Time
[0078] Cells, including hematopoietic cells, comprise cell cycle
control mechanisms, which include cyclins and cyclin-dependent
kinases (CDKs), that control the rate of cell division. Cell cycle
checkpoints are used by cells to monitor and regulate the progress
of the cell cycle. If a cell fails to meet the requirements of a
phase it will not be allowed to proceed to the next phase until the
requirements have been met. The processes associated with
qualifying the cell for progression through the different phases of
the cell cycle (checkpoint regulation) are relatively slow and
contribute to the relatively modest rate of cell division observed
in mammalian cells, even under optimal in vitro culture
conditions.
[0079] In one embodiment of the method of producing erythrocytes,
the method uses hematopoietic cells that have a reduced population
doubling time. In a specific embodiment, the hematopoietic cells
are modified to express higher-than-normal levels of a cell cycle
activator, or a lower-than-normal level of a cell cycle inhibitor,
wherein the engineered cells have a detectably shorter doubling
time than unmodified hematopoietic cells. In a more specific
embodiment, the hematopoietic cells are modified to express a
higher-than-normal level of one or more of the cell cycle activator
cyclin T2 (CCNT2), cyclin T2B (CCNT2B), CDC7L1, CCN1, cyclin G
(CCNG2), cyclin H (CCNH), CDKN2C, CDKN2D, CDK4, cyclin D1, cyclin
A, cyclin B, Hes1, Hox genes and/or FoxO.
[0080] In another more specific embodiment, the hematopoietic cells
express a lower-than-normal level of CDK inhibitors p21, p27 and/or
TReP-132. Reduction of expression of CDK inhibitors can be
accomplished by any means known in the art, e.g., the use of small
molecule inhibitors, antisense oligonucleotides targeted to a p21,
p27 and/or TReP-132 DNA, pre-mRNA or mRNA sequence, RNAi, or the
like.
[0081] Modifications of hematopoietic progenitor cells in the
context of the present method of producing erythrocytes are
expected to be safe in a therapeutic context, as erythrocytes are
enucleated and incapable of replication.
[0082] In another specific embodiment, the hematopoietic cells are
modified to express higher-than-normal levels of a cell cycle
activator, wherein the engineered cells have a detectably shorter
doubling time than, or detectably increased rate of proliferation
compared to, unmodified hematopoietic cells, and where the
increased expression of a cell cycle activator is inducible. Any
inducible promoter known in the art can be used to construct such a
modified hematopoietic cell, e.g., a tetracycline-inducible gene
expression system using a stably expressed reverse
tetracycline-controlled transactivator (rtTA) under the control of
a CMV promoter (e.g., REVTET-ON.RTM. System, Clontech Laboratories,
Palo Alto, Calif.); U.S. Patent Application Publication No.
2007/0166366 "Autologous Upregulation Mechanism Allowing Optimized
Cell Type-Specific and Regulated Gene Expression Cells"; and U.S.
Patent Application Publication No. 2007/0122880 "Vector for the
Inducible Expression of Gene Sequences," the disclosure of each of
which is incorporated herein by reference in its entirety.
[0083] Expression of a gene encoding a cell cycle inhibitor or
negative cell cycle regulator can be disrupted in a hematopoietic
cell, e.g., by homologous or non-homologous recombination using
standard methods. Disruption of expression of a cell cycle
inhibitor or negative cell cycle regulator can also be effected,
e.g., using an antisense molecule to, e.g., p21, p27 and/or
TReP-132.
[0084] In another embodiment, hematopoietic cells used to produce
erythrocytes are modified to express notch 1 ligand such that
expression of the notch 1 ligand results in detectably decreased
senescence of the hematopoietic cells compared to unmodified
hematopoietic cells; see Berstein et al., U.S. Patent Application
Publication 2004/0067583 "Methods for Immortalizing Cells," the
disclosure of which is incorporated herein by reference in its
entirety.
[0085] In another specific embodiment, the medium in which the
hematopoietic cells are expanded enhance faithful DNA replication,
e.g., the medium includes one or more antioxidants.
[0086] In a preferred embodiment, the method of producing
erythrocytes includes a step that excludes any modified
hematopoietic cells, or pre-erythrocyte precursors, from the final
population of isolated erythrocytes produced in the method
disclosed herein. Such separation can be accomplished as described
elsewhere herein on the basis of one or more markers characteristic
of hematopoietic cells not fully differentiated into erythrocytes.
The exclusion step can be performed subsequent to an isolation step
in which erythrocytes are selected on the basis of
erythrocyte-specific markers, e.g., CD36 and/or glycophorin A.
[0087] 5.2.2. Feeder Cell-Independent Expansion of Hematopoietic
Cells
[0088] In certain embodiments, hematopoietic cells used in the
methods provided herein are expanded in culture without the use of
a feeder layer to produce a population of expanded hematopoietic
cells to produce, e.g., a first expanded hematopoietic cell
population. The hematopoietic cells can be expanded by any feeder
cell-independent method known to those of skill in the art. In one
embodiment, feeder-free expansion of hematopoietic cells is the
first of at least two expansion steps prior to differentiation of
the hematopoietic cells into erythrocytes.
[0089] Feeder cell-independent expansion of hematopoietic cells can
take place in any container compatible with cell culture and
expansion, e.g., flask, tube, beaker, dish, multiwell plate, bag or
the like. In a specific embodiment, feeder cell-independent
expansion of hematopoietic cells takes place in a bag, e.g., a
flexible, gas-permeable fluorocarbon culture bag (for example, from
American Fluoroseal). In a specific embodiment, the container in
which the hematopoietic cells are expanded is suitable for
shipping, e.g., to a site such as a hospital or military zone
wherein the expanded hematopoietic cells are further expanded and
differentiated, e.g., using the bioreactor described below.
[0090] Hematopoietic cells, in certain embodiments, are expanded in
a culture medium comprising one or more cytokines or growth
factors. In one embodiment, the medium in which the hematopoietic
cells are expanded comprise one or more of Flt-3 ligand,
thrombopoietin, and stem cell factor (SCF). In a specific
embodiment, hematopoietic cells at about 2.times.10.sup.4 cells per
milliliter are expanded in contact with about 50 ng/mL Flt-3
ligand, about 100 ng/mL thrombopoietin, and about 100 ng/mL SCF.
Expansion times can range, e.g., from about 3 days to about 21
days, e.g., about 7 days.
[0091] In one embodiment, hematopoietic cells are expanded by
culturing said cells in contact with an immunomodulatory compound,
e.g., a TNF-.alpha. inhibitory compound, for a time and in an
amount sufficient to cause a detectable increase in the
proliferation of the hematopoietic cells over a given time,
compared to an equivalent number of hematopoietic cells not
contacted with the immunomodulatory compound. See, e.g., U.S.
Patent Application Publication No. 2003/0235909, the disclosure of
which is hereby incorporated by reference in its entirety. In a
preferred embodiment, the immunomodulatory compound is
3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione;
3-(4'aminoisolindoline-1'-one)-1-piperidine-2,6-dione;
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione;
4-amino-2-[(3RS)-2,6-dioxopiperidin-3-yl]-2H-isoindole-1,3-dione;
.alpha.-(3-aminophthalimido) glutarimide; pomalidomide,
lenalidomide, or thalidomide. In another embodiment, said
immunomodulatory compound is a compound having the structure
##STR00001##
wherein one of X and Y is C.dbd.O, the other of X and Y is C.dbd.O
or CH.sub.2, and R.sup.2 is hydrogen or lower alkyl, or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
enantiomer, diastereomer, racemate, or mixture of stereoisomers
thereof. In another embodiment, said immunomodulatory compound is a
compound having the structure
##STR00002##
[0092] wherein one of X and Y is C.dbd.O and the other is CH.sub.2
or C.dbd.O;
[0093] R.sup.1 is H, (C.sub.1-C.sub.8)alkyl,
(C.sub.3-C.sub.7)cycloalkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, benzyl, aryl,
(C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl,
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl, C(O)R.sup.3,
C(S)R.sup.3, C(O)OR.sup.4, (C.sub.1-C.sub.8)alkyl-N(R.sup.6).sub.2,
(C.sub.1-C.sub.8)alkyl-OR.sup.5,
(C.sub.1-C.sub.8)alkyl-C(O)OR.sup.5, C(O)NHR.sup.3, C(S)NHR.sup.3,
C(O)NR.sup.3R.sup.3', C(S)NR.sup.3R.sup.3' or
(C.sub.1-C.sub.8)alkyl-O(CO)R.sup.5;
[0094] R.sup.2 is H, F, benzyl, (C.sub.1-C.sub.8)alkyl,
(C.sub.2-C.sub.8)alkenyl, or (C.sub.2-C.sub.8)alkynyl;
[0095] R.sup.3 and R.sup.3' are independently
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.7)cycloalkyl,
(C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, benzyl, aryl,
(C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl,
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl,
(C.sub.0-C.sub.8)alkyl-N(R.sup.6).sub.2,
(C.sub.1-C.sub.8)alkyl-OR.sup.5,
(C.sub.1-C.sub.8)alkyl-C(O)OR.sup.5,
(C.sub.1-C.sub.8)alkyl-O(CO)R.sup.5, or C(O)OR.sup.5;
[0096] R.sup.4 is (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, (C.sub.1-C.sub.4)alkyl-OR.sup.5, benzyl,
aryl, (C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl, or
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl;
[0097] R.sup.5 is (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, benzyl, aryl, or
(C.sub.2-C.sub.5)heteroaryl;
[0098] each occurrence of R.sup.6 is independently H,
(C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, benzyl, aryl,
(C.sub.2-C.sub.5)heteroaryl, or
(C.sub.0-C.sub.8)alkyl-C(O)O--R.sup.5 or the R.sup.6 groups can
join to form a heterocycloalkyl group;
[0099] n is 0 or 1; and
[0100] * represents a chiral-carbon center;
[0101] or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, enantiomer, diastereomer, racemate, or mixture of
stereoisomers thereof. In another embodiment, said immunomodulatory
compound is a compound having the structure
##STR00003##
[0102] wherein:
[0103] one of X and Y is C.dbd.O and the other is CH.sub.2 or
C.dbd.O;
[0104] R is H or CH.sub.2OCOR';
[0105] (i) each of R.sup.1, R.sup.2, R.sup.3, or R.sup.4,
independently of the others, is halo, alkyl of 1 to 4 carbon atoms,
or alkoxy of 1 to 4 carbon atoms or (ii) one of R.sup.1, R.sup.2,
R.sup.3, or R.sup.4 is nitro or --NHR.sup.5 and the remaining of
R.sup.1, R.sup.2, R.sup.3, or R.sup.4 are hydrogen;
[0106] R.sup.5 is hydrogen or alkyl of 1 to 8 carbons
[0107] R.sup.6 hydrogen, alkyl of 1 to 8 carbon atoms, benzo,
chloro, or fluoro;
[0108] R' is R.sup.7--CHR.sup.10--N(R.sup.8R.sup.9);
[0109] R.sup.7 is m-phenylene or p-phenylene or
--(C.sub.nH.sub.2n)-- in which n has a value of 0 to 4;
[0110] each of R.sup.8 and R.sup.9 taken independently of the other
is hydrogen or alkyl of 1 to 8 carbon atoms, or R.sup.8 and R.sup.9
taken together are tetramethylene, pentamethylene, hexamethylene,
or --CH.sub.2CH.sub.2X.sub.1CH.sub.2CH.sub.2-- in which X.sub.1 is
--O--, --S--, or --NH--;
[0111] R.sup.10 is hydrogen, alkyl of to 8 carbon atoms, or phenyl;
and
[0112] * represents a chiral-carbon center;
or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,
enantiomer, diastereomer, racemate, or mixture of stereoisomers
thereof.
[0113] In a specific, preferred embodiment, expansion of
hematopoietic cells is performed in IMDM supplemented with 20% BITS
(BSA, recombinant human insulin and transferrin), SCF, Flt-3
ligand, IL-3, and
4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (10
.mu.M in 0.05% DMSO). In a more specific embodiment, about
5.times.10.sup.7 hematopoietic cells, e.g., CD34.sup.+ cells, are
expanded in the medium to from about 5.times.10.sup.10 cells to
about 5.times.10.sup.12 cells, which are resuspended in 100 mL of
IMDM to produce a population of expanded hematopoietic cells. The
population of expanded hematopoietic cells is preferably
cryopreserved to facilitate shipping.
[0114] The expanded hematopoietic cells produced without feeder
cells as described above, e.g., the first expanded hematopoietic
cell population, can be further expanded using feeder cells to
produce a second expanded hematopoietic cell population, as
described in the following Section.
[0115] 5.2.3. Expansion Using Feeder Layers
[0116] In another embodiment, the hematopoietic cells used in the
methods provided herein, e.g., a first expanded hematopoietic cell
population produced by non-feeder cell-dependent expansion of
hematopoietic cells as described above, can be cultured and
expanded using a layer of feeder cells, e.g., to produce a second
expanded hematopoietic cell population. In one embodiment,
expansion of the hematopoietic cells in the presence of a feeder
cell layer is the second of at least two hematopoietic cell
expansion steps prior to differentiation of the hematopoietic cells
to erythrocytes. Though not wishing to be bound by theory,
expansion with feeder cells is expected to be "asymmetric," that
is, expansion produces a combination of expansion of relatively
undifferentiated hematopoietic cells and relatively more
differentiated, lineage-committed cells. Lineage-committed cells
can, in certain embodiments, be removed during the expansion
phase.
[0117] The feeder cells can be any feeder cells used or known to be
useful in the art. Feeder cells can be of the same species as the
hematopoietic cells, or of a different species. In certain
embodiments, feeder cells are from the same individual as the
hematopoietic cells, e.g., the feeder cells and hematopoietic cells
are matched. Feeder cells, like the hematopoietic cells, can be
autologous to a particular recipient. Feeder cell types useful for
culturing hematopoietic cells can be, e.g., bone marrow-derived
mesenchymal stem cells (e.g., see U.S. Pat. No. 5,486,359), tissue
culture plastic-adherent placental stem cells, mesenchymal-like
stem cells from cord blood, placental blood or peripheral blood,
adult stem cells, or the like). In another embodiment, the feeder
cells are fully-differentiated cells, e.g., fibroblasts, such as
human skin fibroblasts or mouse embryonic fibroblasts, or human
umbilical vein endothelial cells.
[0118] In certain embodiments, the feeder cells are not treated to
reduce proliferation or differentiation. In certain other
embodiments, the feeder layer cells are treated with, e.g.,
mitomycin C or ionizing radiation, e.g., gamma radiation, to
suppress or prevent proliferation of the feeder cells, e.g., about
100 cGy to about 100 Gray, e.g., 1 Gray to about 60 Gray. In a
specific embodiment, the cells are treated with 1500 cGy ionizing
radiation to produce feeder cells. Methods of irradiating feeder
cells can be found in, e.g., U.S. Application Publication No.
2006/0223183. In a specific embodiment, the feeder cells are
cryopreserved prior to irradiation. In another specific embodiment,
a plurality of human fibroblasts are irradiated with from about 1
Gray to about 60 Gray, e.g., about 1.5 Gray, ionizing radiation to
produce a plurality of feeder cells.
[0119] In one embodiment, the feeder cells and hematopoietic cells
are cultured in a bioreactor, e.g., a bioreactor substantially as
described elsewhere herein. In a specific embodiment of the
bioreactor, the hematopoietic cells and feeder cells are
co-cultured, that is, such that a plurality of the hematopoietic
cells are in physical contact with a plurality of the feeder cells.
In another embodiment, the hematopoietic cells are cultured
separately, wherein culture medium from the feeder cells is
circulated to the hematopoietic cells. In a specific embodiment,
the hematopoietic cells expanded using a feeder layer are
hematopoietic cells that were expanded with an immunomodulatory
compound prior to culture with or contact with said feeder
layer.
[0120] 5.2.4. Adherent Placental Stem Cells as Feeder Cells
[0121] As noted above, adherent placental stem cells can be used as
feeder cells in the methods provided herein. Adherent placental
stem cells are described in, and are obtainable by the methods
disclosed in, U.S. Pat. Nos. 7,045,148 and 7,255,879, and in United
States Patent Application Publication Nos. 2007/0275362, and
2008/0032401, the disclosures of each of which are incorporated
herein in their entireties.
[0122] Adherent placental stem cells useful in the methods provided
herein express a plurality of markers that can be used to identify
and/or isolate the stem cells, or populations of cells that
comprise the stem cells. The adherent placental stem cells, and
stem cell populations thereof (that is, two or more placental stem
cells) include stem cells and stem cell-containing cell populations
obtained directly from the placenta, or any part thereof (e.g.,
amnion, chorion, placental cotyledons, and the like). Adherent
placental stem cell populations also includes populations of (that
is, two or more) placental stem cells in culture, and a population
in a container, e.g., a bag. The adherent placental stem cells
described herein as usable as feeder layers for hematopoietic cell
expansion are not trophoblasts, cytotrophoblasts, embryonic stem
cells or embryonic germ cells.
[0123] The adherent placental stem cells described herein generally
express the markers CD73, CD105, CD200, HLA-G, and/or OCT-4, and do
not express CD34, CD38, or CD45. Placental stem cells can also
express HLA-ABC (MHC-1) and HLA-DR. These markers can be used to
identify adherent placental stem cells, and to distinguish adherent
placental stem cells from other stem cell types. Because the
placental stem cells can express CD73 and CD105, they can have
mesenchymal stem cell-like characteristics. However, because the
placental stem cells can express CD200 and HLA-G, a fetal-specific
marker, they can be distinguished from mesenchymal stem cells,
e.g., bone marrow-derived mesenchymal stem cells, which express
neither CD200 nor HLA-G. In the same manner, the lack of expression
of CD34, CD38 and/or CD45 identifies the placental stem cells as
non-hematopoietic stem cells.
[0124] Thus, in one embodiment, the feeder cells are isolated
adherent placental stem cells that are CD200.sup.+ or HLA-G.sup.+.
In another embodiment, the feeder cells are a population of cells
comprising, e.g., that is enriched for, adherent CD200.sup.+,
HLA-G.sup.+ placental stem cells. In another embodiment, the feeder
cells are isolated adherent placental stem cells that are
CD73.sup.+, CD105.sup.+, and CD200.sup.+. In another embodiment,
the feeder cells are an isolated population of cells comprising,
e.g., that is enriched for, adherent CD73.sup.+, CD105.sup.+,
CD200.sup.+ placental stem cells. In another embodiment, the feeder
cells are isolated adherent placental stem cells that are
CD200.sup.+ and OCT-4.sup.+. In another embodiment, the feeder
cells are an isolated population of cells comprising, e.g., that is
enriched for, adherent CD200.sup.+, OCT-4.sup.+ placental stem
cells. In another embodiment, the feeder cells are isolated
adherent placental stem cells that are CD73.sup.+, CD105.sup.+ and
HLA-G.sup.+. In another embodiment, the feeder cells are an
isolated population of cells comprising, e.g., that is enriched
for, adherent CD73.sup.+, CD105.sup.+ and HLA-G.sup.+ placental
stem cells. In another embodiment, the feeder cells are isolated
adherent placental stem cells that are CD73.sup.+ and CD105.sup.+
and which facilitate the formation of one or more embryoid-like
bodies in a population of isolated placental cells comprising said
stem cells when said population is cultured under conditions that
allow formation of embryoid-like bodies. In another embodiment, the
feeder cells are a population of isolated cells comprising, e.g.,
that is enriched for, adherent CD73.sup.+, CD105.sup.+ placental
stem cells, wherein said population forms one or more embryoid-like
bodies under conditions that allow formation of embryoid-like
bodies. In another embodiment, the feeder cells are isolated
adherent placental stem cells that are OCT-4.sup.+ and which
facilitate formation of one or more embryoid-like bodies in a
population of isolated placental cells comprising said stem cell
when cultured under conditions that allow formation of
embryoid-like bodies. In another embodiment, the feeder cells are a
population of isolated cells comprising, e.g., that is enriched
for, adherent OCT-4.sup.+ placental stem cells, wherein said
population forms one or more embryoid-like bodies when cultured
under conditions that allow the formation of embryoid-like
bodies.
[0125] In another embodiment, the feeder cells comprise isolated
adherent placental stem cells that are CD10.sup.+, CD34.sup.-,
CD105.sup.+, and CD200.sup.+. In a specific embodiment of the above
embodiments, said stem cells are additionally CD90.sup.+ and
CD45.sup.-. In another embodiment, the feeder cells are isolated
adherent placental stem cells that are HLA-A,B,C.sup.-, CD45.sup.-,
CD133.sup.- and CD34.sup.-. In another embodiment, the feeder cells
are isolated adherent placental stem cell that is CD10.sup.+,
CD13.sup.+, CD33.sup.+, CD45.sup.-, CD117.sup.- and CD133.sup.-. In
another embodiment, the feeder cells are isolated adherent
placental stem cells that are CD10.sup.-, CD33.sup.-, CD44.sup.+,
CD45.sup.-, and CD117.sup.-. In another embodiment, the feeder
cells are isolated adherent placental stem cells that are HLA
A,B,C.sup.-, CD45.sup.-, CD34.sup.-, CD133.sup.-, positive for
CD10, CD13, CD38, CD44, CD90, CD105, CD200 and/or HLA-G, and/or
negative for CD117. In another embodiment, feeder cells are
isolated adherent placental stem cells that are CD200.sup.+ and
CD10.sup.+, as determined by antibody binding, and CD117.sup.-, as
determined by both antibody binding and RT-PCR. In another
embodiment, the feeder cells are isolated adherent placental stem
cells that are CD10.sup.+, CD29.sup.-, CD54.sup.+, CD200.sup.+,
HLA-G.sup.+, HLA class I.sup.+ and
.beta.-2-microglobulin.sup.+.
[0126] Adherent placental stem cells used as feeder layers can be
freshly isolated from the placenta, or can have been, for example,
passaged at least once, at least three times, at least five times,
at least 10 times, at least 15 times, or at least 20 times.
[0127] Adherent placental stem cells can be used as feeder cells,
for example, by contacting adherent placental stem cells with
mitomycin C, or irradiating the cells, for a time sufficient to
detectably suppress proliferation of the cells, and plating the
cells, e.g., on a tissue culture surface, at about 5,000 cells per
cm.sup.2. The adherent placental stem cells are allowed to attach
to the surface, e.g., for 10 minutes to 60 minutes. The plated,
attached adherent placental stem cells are then ready to be
inoculated with hematopoietic cells for hematopoietic cell culture
and expansion.
[0128] 5.3. Production of Erythrocytes from Hematopoietic Cells
[0129] Production of erythrocytes from hematopoietic cells, e.g.,
the expanded hematopoietic cells described above, is preferably
performed in a bioreactor, e.g., the bioreactor exemplified
elsewhere herein.
[0130] Differentiation of hematopoietic cells into erythrocytes
and/or polychromatophilic erythrocytes can be accomplished by
contacting the hematopoietic cells with one or more erythrogenic
cytokines and/or growth factors. In one embodiment, hematopoietic
cells are contacted with one or more of stem cell factor (SCF),
erythropoietin (Epo), insulin-like growth factor (IGF-1), FMS-like
tyrosine kinase 3 ligand (Flt3-L), and thrombopoietin (Tpo) in an
amount and for a time sufficient to cause the differentiation of a
plurality of the hematopoietic cells to erythrocytes and/or
polychromatophilic erythrocytes. In various specific embodiments,
at least 50%, 55%, 60%, 65%, 70%. 75%, 80%, 85%, 90%, 95%, 97%,
98%, or 99% of the hematopoietic cells are differentiated to
erythrocytes and/or polychromatophilic erythrocytes.
[0131] In a specific embodiment, the hematopoietic cells are
contacted with cytokines, e.g., SCF, IGF-1 and erythropoietin in an
amount and for a time sufficient to cause differentiation of a
plurality of the hematopoietic cells into erythrocytes and/or
polychromatophilic erythrocytes. In certain embodiments, the
hematopoietic cells are contacted with one or more cytokines or
growth factors in an amount and for a time sufficient to cause
differentiation of a plurality of the hematopoietic cells into
erythrocytes and polychromatophilic erythrocytes (also known as
reticulocytes, blood reticulocytes, diffusely basophilic
erythrocytes, or marrow reticulocytes). In certain other
embodiments, the hematopoietic cells are contacted with one or more
cytokines or growth factors in an amount and for a time sufficient
to cause differentiation of a plurality of the hematopoietic cells
into a precursor of an erythrocyte, e.g., into colony-forming
units-granulocyte, erythrocyte, monocyte; blast-forming
units-erythrocyte; colony-forming units-erythrocyte,
pronormoblasts, basophilic normoblasts, polychromatic normoblasts,
orthochromic normoblasts, or polychromatic erythrocytes
(reticulocytes).
[0132] The above cytokines can be used in any amount that causes
differentiation a plurality of the hematopoietic cells into
erythrocytes. Typical amounts of cytokines used, for hematopoietic
cells expanded for about 7 days from a starting population of,
e.g., about 1.times.10.sup.4 to 2.times.10.sup.4 hematopoietic
cells per milliliter, are about 50 ng/mL SCF; about 3 units/mL Epo;
50 ng/mL IGF-1; 50 ng/mL Flt3-L; and/or 100 ng/mL Tpo in, e.g.,
RPMI medium. Differentiation of the hematopoietic cells in, e.g.
SCF, IGF-1 and Epo can proceed for, e.g., 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, typically about
14 days. Optionally, the differentiated cells can be collected,
resuspended to about 5.times.10.sup.5 per milliliter in medium,
e.g., RPMI medium comprising Epo (e.g., at about 3 U/mL), IGF-1
(e.g., at about 20 ng/ml), IL-11 (e.g., at about 20 ng/ml) and IL-3
(e.g., at about 50 ng/ml), and cultured for an additional 1, 2, 3,
4, 5, 6, 7, 8 9 or 10 days, typically about 6 days.
[0133] In one embodiment, differentiation of hematopoietic cells,
e.g., the expanded hematopoietic cells described above, can be
accomplished by culturing said cells in contact with an
immunomodulatory compound, e.g., a TNF-.alpha. inhibitory compound
as described above, for a time and in an amount sufficient to cause
a detectable increase in the proliferation of the hematopoietic
cells over a given time, compared to an equivalent number of
hematopoietic cells not contacted with the immunomodulatory
compound. See, e.g., U.S. Patent Application Publication No.
2003/0235909, the disclosure of which is incorporated herein by
reference in its entirety.
[0134] Differentiation of the hematopoietic cells into erythrocytes
can be assessed by detecting erythrocyte-specific markers, e.g., by
flow cytometry. Erythrocyte-specific markers include, but are not
limited to, CD36 and glycophorin A. Differentiation can also be
assessed by visual inspection of the cells under a microscope. The
presence of typical biconcave cells confirms the presence of
erythrocytes. The presence of erythrocytes (including
reticulocytes) can be confirmed using a stain for deoxyribonucleic
acid (DNA), such as Hoechst 33342, TO-PRO.RTM.-3, or the like.
Nucleated precursors to erythrocytes typically stain positive with
a DNA-detecting stain, while erythrocytes and reticulocytes are
typically negative. Differentiation of hematopoietic cells to
erythrocytes can also be assessed by progressive loss of
transferring receptor (CD71) expression and/or laser dye styryl
staining during differentiation. Erythrocytes can also be tested
for deformability using, e.g., an ektacytometer or diffractometer.
See, e.g., Bessis M and Mohandas N, "A Diffractometric Method for
the Measurement of Cellular Deformability," Blood Cells 1:307
(1975); Mohandas N. et al., "Analysis of Factors Regulating
Erythrocyte Deformability," J. Clin. Invest. 66:563 (1980); Groner
W et al., "New Optical Technique for Measuring Erythrocyte
Deformability with the Ektacytometer," Clin. Chem. 26:1435 (1980).
Fully-differentiated erythrocytes have a mean corpuscular volume
(MCV) of about 80 to about 108 fL (femtoliters); mean corpuscular
hemoglobin (MCH) of about 17 to about 31 pg, and a mean corpuscular
hemoglobin concentration (MCHC) of about 23% to about 36%.
[0135] 5.4. Separation of Erythrocytes from Precursors
[0136] Erythrocytes produced by the methods described above are
preferably separated from hematopoietic cells, and, in certain
embodiments, from precursors of erythrocytes. Such separation can
be effected, e.g., using antibodies to CD36 and/or glycophorin A.
Separation can be achieved by known methods, e.g.,
antibody-mediated magnetic bead separation, fluorescence-activated
cell sorting, passage of the cells across a surface or column
comprising antibodies to CD36 and/or glycophorin A, or the like. In
another embodiment, erythrocyte separation is achieved by
deoxygenating the culture medium comprising the erythrocytes,
followed by magnetic separation of deoxygenated erythrocytes from
other cells.
[0137] Erythrocytes can be continuously separated from a population
of cells, e.g., from a second expanded hematopoietic cell
population as described above, or can be separated at intervals. In
certain embodiments, for example, isolation of erythrocytes is
performed, e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,
30, 35, 40, 45, 50, 55 or 60 minutes, or every 1, 1.5, 2, 2.5, 3,
3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,
10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or
24 hours, or more, or when one or more culture condition criteria
are met, e.g., achievement in the culture of a particular cell
density; achievement in the culture of a particular number of cells
per milliliter expressing certain erythrocyte markers, e.g., CD36
or glycophorin A; or the like. Separation of erythrocytes from a
cell population is preferably performed using a bioreactor, as
described below.
[0138] 5.5. Bioreactor Production of Erythrocytes
[0139] In another aspect of the method of producing erythrocytes,
hematopoietic cells are expanded using a bioreactor. In one
embodiment, hematopoietic cells are expanded in a bioreactor in the
presence of feeder cells. In another embodiment, hematopoietic
cells are differentiated in a bioreactor. In a more preferred
embodiment, the hematopoietic cells are expanded in a bioreactor,
e.g., in the presence of feeder cells, then differentiated in the
bioreactor. The bioreactor in which the hematopoietic cells are
differentiated can be the same bioreactor in which the
hematopoietic cells are expanded, or can be a separate bioreactor.
In another embodiment, the bioreactor is constructed to facilitate
expansion of the hematopoietic cells entirely in the bioreactor. In
another embodiment, the bioreactor is constructed to allow
expansion of hematopoietic cells in conjunction with feeder cells.
In another embodiment, the bioreactor is constructed so as to
physically separate the hematopoietic cells from the feeder cells.
In another embodiment, the bioreactor is constructed to allow the
hematopoietic cells and feeder cells to contact one another.
[0140] In another embodiment, the bioreactor is constructed to
allow continuous flow of cells in media, enabling the continuous
separation of differentiated erythrocytes from remaining cells in
the bioreactor. The continuous flow and cell separation allows for
the bioreactor to be constructed in a substantially smaller volume
than would bioreactors using batch methods of producing
erythrocytes. In another embodiment, the bioreactor is constructed
to allow periodic, e.g., non-continuous flow of cells in media,
enabling the periodic separation of differentiated erythrocytes
from remaining cells in the bioreactor. The periodic flow and cell
separation preferably allows for the bioreactor to be constructed
in a substantially smaller volume than would bioreactors using
batch methods of producing erythrocytes. In specific embodiments,
isolation of erythrocytes is performed, e.g., every 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes,
or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5,
8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23 or 24 hours, or more. In another specific
embodiment, isolation of erythrocytes is performed periodically
when one or more culture condition criteria are met, e.g.,
achievement in the culture of a particular cell density;
achievement in the culture of a particular number of cells per
milliliter expressing certain erythrocyte markers, e.g., CD36 or
glycophorin A; or the like.
[0141] In certain embodiments, the bioreactor is disposable.
[0142] In one embodiment, the bioreactor comprises a culturing
element and a cell separation element. In another embodiment, the
bioreactor comprises a medium gas provision element. In another
embodiment, the bioreactor comprises a cell factor element
comprising one or more bioactive compounds. In another embodiment,
the elements of the bioreactor are modular; e.g., separable from
each other and/or independently usable. In one embodiment, the
capacity of the bioreactor is about 100 mL, 200 mL, 300 mL, 400 mL,
500 mL, 600 mL, 700 mL, 800 mL, or about 900 mL, or about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45 or 50
liters. In another embodiment, the bioreactor, including all
components, occupies about 47 cubic feet or less. In another
embodiment, the bioreactor is capable of culturing up to about
10.sup.10, 10.sup.11, or about 10.sup.12 cells, e.g., hematopoietic
cells.
[0143] In one embodiment, the culturing element comprises a
compartment able to receive culture medium, e.g., culture medium
comprising hematopoietic cells or culture medium comprising feeder
cells. The culturing element comprises a port that allows for the
introduction of media and/or hematopoietic cells for culture. Such
a port can be any art-acceptable port for such devices, e.g., a
Luer-lock seal port. The culturing element also comprises one or
more ports for the passage of media to the cell separation element.
The culturing element optionally further comprises a port for the
introduction of bioactive compounds into the interior of the
culturing element, e.g., a port that facilitates connection of the
cell factor element to the culturing element. In a specific
embodiment, hematopoietic cells, including differentiating
hematopoietic cells, in the culturing element are continuously
circulated in medium to a cell separation element (see below) to
isolate erythrocytes and/or polychromatophilic erythrocytes and/or
other erythrocyte precursors.
[0144] The culturing element, in a specific embodiment, comprises a
plurality of interior surfaces or structures suitable for the
culture of feeder cells, e.g., stromal cells or adherent placental
stem cells. Such surfaces can be, e.g., tubes, cylinders, hollow
fibers, a porous substrate, or the like. The surfaces can be
constructed of any material suitable for the culture of cells,
e.g., tissue culture plastic, flexible pharmaceutical grade
plastic, hydroxyapatite, polylactic acid (PLA), polyglycolic acid
copolymer (PLGA), polyurethane, polyhydroxyethyl methacrylate, or
the like. Hollow fibers typically range from about 100 .mu.m to
about 1000 .mu.m in diameter, and typically comprise pores that
allow passage of molecules no more than about 5 kDa, 10 kDa, 15
kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90
kDa, 100 kDa, 125 kDa, 150 kDa, 175 kDa, 200 kDa, 150 kDa, 300 kDa,
350 kDa, 400 kDa, 450 or 500 kDa.
[0145] In one embodiment, the surfaces on which the feeder cells
are cultured physically separate the feeder cells from the
hematopoietic cells. For example, the surfaces can be hollow fibers
in the lumen of which the feeder cells are grown, while
hematopoietic cells are cultured in media surrounding the fibers.
The surfaces can also comprise a set of stacked membranes that
separate two medium compartments, one for the feeder layers and
another for the hematopoietic cells. In another embodiment, the
surface on which the feeder cells are grown allows for the direct
contact between feeder cells and hematopoietic cells.
[0146] The cell separation element comprises at least one port for
receiving medium, comprising cells, from the culturing element. The
cell separation element comprises one or more components that
facilitate or enable the separation of at least one type of cell,
e.g., erythrocytes, from cells in medium from the culture element.
Such separation can be effected, e.g., using antibodies to CD36
and/or glycophorin A. Separation can be achieved by known methods,
e.g., antibody-mediated magnetic bead separation,
fluorescence-activated cell sorting, passage of the cells across a
surface or column comprising antibodies to CD36 and/or glycophorin
A, or the like. In a specific embodiment, the cell separation
element is connected to the cell culturing element, and medium
comprising hematopoietic cells, differentiating hematopoietic cells
and erythrocytes is continually passed through the cell separation
element so as to continually remove cells, e.g., erythrocytes from
the medium.
[0147] In another embodiment, erythrocyte separation is achieved by
deoxygenating culture medium comprising the erythrocytes, followed
by magnetic attraction of deoxygenated erythrocytes, e.g., to a
surface or other point of collection.
[0148] In another embodiment, the bioreactor comprises a cell
separation element. The cell separation element can comprise one or
more components that enable the separation of one or more
non-erythrocytic cells (e.g., undifferentiated or non-terminally
differentiated hematopoietic cells) from erythrocytes in the
medium. In certain embodiments, the cell separation element is able
to calculate an approximate number of erythrocytes generated, or is
able to alert a user that a sufficient number of erythrocytes has
been generated to constitute a unit, according to preset user
parameters.
[0149] The bioreactor, in another embodiment, further comprises a
gas provision element that provides appropriate gases to the
culture environment, e.g., contacts the culture medium with a
mixture of 80% air, 15% O.sub.2 and 5% CO.sub.2, 5% CO.sub.2 in
air, or the like. In another embodiment, the bioreactor comprises a
temperature element that maintains the medium, the bioreactor, or
both at a substantially constant temperature, e.g., about
35.degree. C. to about 39.degree. C., or about 37.degree. C. In
another embodiment, the bioreactor comprises a pH monitoring
element that maintains the medium at a constant pH, e.g., about pH
7.2 to about pH 7.6, or about pH 7.4. In specific embodiments, the
temperature element and/or pH monitoring element comprises a
warning that activates when temperature and/or pH exceed or fall
below set parameters. In other specific embodiments, the
temperature element and/or pH monitoring element are capable of
correcting out-of-range temperature and/or pH.
[0150] In a specific embodiment, the bioreactor comprises a cell
separation element and a gas provision element that provides gases
to the culture environment, whereby the gas provision element
enables the partial or complete deoxygenation of erythrocytes,
enabling erythrocyte separation based on the magnetic properties of
the hemoglobin contained therein. In a more specific aspect, the
bioreactor comprises an element that allows for the regular, or
iterative, deoxygenation of erythrocytes produced in the
bioreactor, to facilitate magnetic collection of the
erythrocytes.
[0151] In another embodiment, the function of the bioreactor is
automated, e.g., controlled by a computer. The computer can be, for
example, a desktop personal computer, a laptop computer, a
Handspring, PALM.RTM. or similar handheld device; a minicomputer,
mainframe computer, or the like.
[0152] 5.6. Erythrocyte Units Produced From Hematopoietic Cells
[0153] Erythrocyte units produced according to the methods detailed
above can comprise erythrocytes in any useful number or combination
of genetic backgrounds.
[0154] In various embodiments, erythrocyte units produced by the
methods provided herein comprise at least about, at most about, or
about 1.times.10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.10, 5.times.10.sup.10,
1.times.10.sup.11, 5.times.10.sup.11 or 1.times.10.sup.12
erythrocytes. In various other embodiments, the erythrocyte units
comprise at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, 98% or 99% completely-differentiated erythrocytes. In
various other embodiments, the erythrocyte units comprise less than
60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% erythrocyte precursors
of any kind. In certain embodiments, the erythrocyte units produced
by the methods described herein comprise less than about 60%, about
50%, about 40%, about 30%, about 20%, about 19%, about 18%, about
17%, about 16%, about 15%, about 14%, about 14%, about 12%, about
11%, about 10%, about 9%, about 8%, about 7%, about 6% or about 5%
reticulocytes, or other non-erythrocytic hematopoietic cells. In
another embodiment, the unit comprises erythrocytes from
hematopoietic cells from a single individual. In another
embodiment, the unit comprises erythrocytes differentiated from
hematopoietic cells from a plurality of individuals. In another
embodiment, the unit comprises erythrocytes from hematopoietic
cells from matched human placental perfusate and cord blood. In
another embodiment, substantially all (e.g., greater than 99%) of
the erythrocytes in a unit of erythrocytes are type O. In another
embodiment, substantially all (e.g., greater than 99%) of the
erythrocytes in a unit of erythrocytes are type A. In another
embodiment, substantially all (e.g., greater than 99%) of the
erythrocytes in a unit of erythrocytes are type B. In another
embodiment, substantially all (e.g., greater than 99%) of the
erythrocytes in a unit of erythrocytes are type AB. In another
embodiment, substantially all (e.g., greater than 99%) of the
erythrocytes in a unit of erythrocytes are Rh positive. In another
embodiment, substantially all (e.g., greater than 99%) of the
erythrocytes in a unit of erythrocytes are Rh negative.
[0155] Naturally-occurring erythrocytes possess certain
characteristics that allow the flow of blood through capillaries.
For example, erythrocytes in the aggregate produce non-Newtonian
flow behavior, e.g., the viscosity of blood is highly dependent
upon shear rates. Normal erythrocytes are deformable and able to
build up aggregates/rouleaux. The deformability of erythrocytes
appears to be related to their lifespan in the blood, about 100-120
days; removal of erythrocytes from the blood appears to be related
to loss of deformability. Normal aggregability of erythrocytes
facilitates the cells' flow through the capillaries, while
abnormally increased or decreased aggregability decreases flow.
Thus, in preferred embodiments, units of erythrocytes produced by
the methods disclosed herein are assayed as a part of quality
control, e.g., for one or more characteristics of
naturally-occurring erythrocytes. In certain embodiments, samples
of erythrocytes produced by the methods disclosed herein are
suspended in natural or artificial plasma and tested for one or
more of viscosity, viscoelasticity, relaxation time, deformability,
aggregability, blood/erythrocyte suspension yield stress, and
mechanical fragility, using normal blood or normal erythrocytes as
a control or comparator. In certain other embodiments, samples of
erythrocytes produced as described herein are assayed for oxygen
carrying capacity and oxygen release capacity, using normal blood
or an equivalent number of naturally-occurring erythrocytes as a
control.
6. EXAMPLES
6.1. Example 1: Phenotypic Characterization of CD34.sup.+ Cells
from Human Placental Perfusate (HPP) and Umbilical Cord Blood
(UCB)
[0156] Umbilical cord blood (UCB) was removed from postpartum
placentas under informed consent. The exsanguinated placentas were
then perfused to generate HPP, as described in U.S. Pat. No.
7,045,148, the disclosure of which is incorporated herein by
reference in its entirety. After removal of red blood cells (RBCs)
the total nucleated cells (TNCs) were collected and frozen. This
method typically resulted in the collection of about
1-2.5.times.10.sup.9 TNCs, compared to around 500 million TNC
isolated from UCB.
[0157] Flow cytometric analysis of the TNC isolated from
exsanguinated placentas indicates a high percentage of CD34.sup.+
cell population as compared to conventional umbilical cord blood
(UCB) generated cellular product. TNC from HPP, collected as above,
contains about 2-6% CD34.sup.+ cells, compared to about 0.3-1% of
the TNC in UCB.
[0158] The flow cytometric analysis of the TNC isolated from HPP
indicates that a high percentage of the CD34.sup.+ cell population
is CD45.sup.- (FIG. 1).
[0159] CD34.sup.+ cells from HPP were plated in a colony-forming
unit assay, and the ratio (%) of the burst forming unit-erythroid
(BFU-E) to the colony forming unit-erythroid (CFU-E) was
determined, as well as the number of colony-forming
unit-granulocyte, macrophage (CFU-GM) and the number of
colony-forming unit-granulocyte, erythrocyte, monocyte (CFU-GEMM)
(Table 1). The clonogenicity was also assessed (Table 1).
TABLE-US-00001 TABLE 1 Cell CFU- Sample Purity BFU-E/CFU-E CFU-GM
GEMM Clonogenicity Donor 1 88% 50.1% 49.5% 0.4% 23.1% Donor 2 92%
54.1% 44.1% 1.7% 26.1% Donor 3 94% 32.7% 60.6% 6.7% 19.7%
[0160] The colony-forming unit assay was performed according to the
manufacturer's protocol (StemCell Technologies, Inc.). In brief,
CD34.sup.+ cell suspensions were placed into a methylcellulose
medium supplemented with stem cell factor (SCF), granulocyte
colony-stimulating factor (G-CSF), granulocyte-macrophage
colony-stimulating factor (GM-CSF), interleukin 3 (IL-3),
interleukin 6 (IL-6) and erythropoietin (Epo) at 100 cells/plate,
300 cells/plate and 1000 cells/plate. For each cell density, a
triplicate assay was performed followed by incubation for 2 to 3
weeks. Colony evaluation and enumeration were performed using light
microscopy.
[0161] In a separate experiment, the ratio (BFU-E)/(CFU-E) for
CD34.sup.+ cells from HPP and UCB was 46% and 30%, respectively
(based on the average value for three donors).
[0162] These results suggest that HPP-derived cells contain a
higher number of CD34.sup.+ cells with increased erythrogenic
activity relative to UCB-derived stem cells.
6.2. Example 2: Expansion of CD34.sup.+ Hematopoietic Cell
Populations
[0163] The CD34.sup.+ cell content of human umbilical cord blood
(UCB) units is often not sufficient to provide for hematopoietic
cell transplants in adult patients. Ex-vivo expansion of CD34.sup.+
cells from UCB is one approach to overcome this CD34.sup.+ cell
dose limitation. This Example demonstrates expansion of CD34.sup.+
cells using a specific immunomodulatory drug,
4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione
(referred to in this Example as pomalidomide).
[0164] The ability of pomalidomide to enhance the expansion of
human UCB derived CD34.sup.+ cells in a short-term serum-free,
cytokine supplemented culture system was evaluated. CD34.sup.+
progenitor cells were enriched from cryopreserved UCB units to
>90% purity and seeded (10.sup.4 CD34.sup.+ cells) in 1 mL of
growth medium, which consists of IMDM plus serum substitute BIT
(BSA, recombinant human insulin and transferrin, 20%), in the
presence of SCF (50 ng/mL), Flt-3 ligand (50 ng/mL), and IL-3 (10
ng/mL). Pomalidomide, dissolved in DMSO, was supplemented at 2.7
.mu.g/mL. The culture was incubated at 37.degree. C., 5% CO.sub.2
for 12 days, with fresh medium added at day 7. Pomalidomide-free
cultures with or without DMSO (0.05% v/v) were used as
controls.
[0165] In one experiment, pomalidomide supplementation resulted in
significantly higher CD34.sup.+ expression in the expanded
population without impacting total nucleated cell expansion
(200-350 fold). CD34.sup.+ phenotype in the pomalidomide-expanded
population was 40-60%, compared with 10-30% in the control.
Additionally, pomalidomide appeared to down-regulate CD38
expression on cultured cells. Pomalidomide-expanded CD34.sup.+
cells were primarily CD38 negative (95%) and expressed lower levels
of CD133 (15% vs. 40% in the control). Pomalidomide-expanded
CD34.sup.+ cells demonstrated substantial improvement in cumulative
colony forming units relative to expanded controls. In another,
similar, experiment, pomalidomide supplementation was confirmed to
result in significantly higher CD34.sup.+ expression in the
expanded population without impacting total nucleated cell
expansion (200-350 fold; FIG. 2A). CD34.sup.+ phenotype in the
pomalidomide-expanded population was 40-60%, compared with 10-30%
in the control (FIG. 2B). Additionally, pomalidomide appeared to
down-regulate CD38 expression on cultured cells.
[0166] Pomalidomide-expanded CD34.sup.+ cells were primarily CD38
negative (97%) and expressed lower levels of CD133 (11.5% vs. 32.3%
in the control). Pomalidomide-expanded CD34.sup.+ cells
demonstrated substantial improvement in cumulative colony forming
units relative to expanded controls.
[0167] The pomalidomide-based CD34.sup.+ expansion process was
scaled up to demonstrate the production of a larger number of
CD34.sup.+ cells. CD34.sup.+ cells were seeded in 10.sup.4/mL
pomalidomide-supplemented medium in a flexible, gas-permeable
fluorocarbon culture bag (American Fluoroseal). After 7 days of
incubation, the culture was centrifuged and exchanged with fresh
pomalidomide-supplemented medium at three times the initial volume.
By day 12, TNC and CD34.sup.+ expansion were 350 (range: 250-700)
and 200 (range: 100-450) fold, respectively (FIG. 3A and FIG. 3B,
respectively). Viability was 86% (range: 80-90%) by trypan blue. A
total of 20 million CD34.sup.+ cells were harvested. These results
demonstrate that pomalidomide significantly enhanced the ex-vivo
expansion of placental derived CD34.sup.+ progenitors and that the
process can produce a sufficient amount of CD34.sup.+ cells for
erythrocytic differentiation (FIG. 3C).
6.3. Example 3: Feeder Cell-Free Expansion of CD34.sup.+ Precursors
and Differentiation into Erythrocytes
[0168] HPP and UCB cells were generally purified using Ficoll to
obtain total nucleated cells (TNCs). TNCs were then used to isolate
CD34.sup.+ cells using anti-CD34 beads and RoboSep following the
protocol provided by the manufacturer (StemCell Technologies,
Inc.). If greater than 80% cell purity was not achieved, further
cell sorting was performed using FACSAria to achieve a purity
greater than 90%. In this experiment, CD34.sup.+ cells were
isolated with about 92% purity.
[0169] In vitro expansion of CD34.sup.+ cells (about
1.times.10.sup.4 CD34.sup.+ cells/mL) was performed in either
Iscove's Modified Dulbecco's Medium (IMDM; GIBCO, Grand Island,
N.Y.) or RPMI Medium (Sigma-Aldrich) containing Flt-3L (50 ng/mL),
SCF (100 ng/mL) and Tpo (100 ng/mL) from day 0 to day 7, and
continued thereafter in IMDM or RPMI medium containing SCF (50
ng/mL), Epo (3 units/mL) and IGF-1 (50 ng/mL) from day 8 to day 14.
Total cell numbers were counted at 8, 9, 12, 15 and 18 days. At day
18 TNC expansion was 300 fold and cell viability was 90%. At day
14, the cells were suspended at 2.times.10.sup.4 to
5.times.10.sup.5 cells/mL in IMDM or RPMI medium containing 3
units/mL Epo, 50 ng/mL IGF-1, 20 ng/mL IL-11 (interleukin 11), and
50 ng/mL IL-3 (interleukin 3) (R&D Systems) and cultured to day
21. FIGS. 4A and 4B show the ex vivo expansion of CD34.sup.+ cells
from HPP (FIG. 4A) and UCB (FIG. 4B) in cytokine supplemented RPMI
Medium.
6.4. Example 4: Expansion of Hematopoietic Cell Populations Using
Feeder Layers
[0170] This Example explains the use of adherent placental stem
cells in co-culturing system using adherent placental stem cells as
a feeder layer.
[0171] To induce erythrocytic differentiation of the expanded
CD34.sup.+ cells, 5.times.10.sup.7 cells were resuspended at 1 to
2.times.10.sup.4/ml and cultured in RPMI Medium containing 50 ng/mL
Flt3-L, 100 ng/mL Tpo, and 100 ng/mL SCF (R&D Systems) for 7
days. The cells collected on day 7 were resuspended at
5.times.10.sup.4 to 1.times.10.sup.5/mL in RPMI medium containing
50 ng/mL SCF, 3 units/mL Epo, and 50 ng/mL IGF-1 (R&D Systems)
and cultured up to 14 days. To further differentiate expanded
CD34.sup.+ cells, the expanded cells collected on day 14 were
resuspended at 2.times.10.sup.4 to 5.times.10.sup.5/mL in RPMI
medium containing 3 units/mL Epo, 50 ng/mL IGF-1 with the presence
of adherent placental stem cells treated with mitomycin C. The
adherent placental stem cells were plated at about 5,000 cells per
cm.sup.2 and allowed to attach to the tissue culture surface.
Aliquots of cultures of CD34.sup.+ cells were taken at different
time points for morphological analysis by Giemsa staining (FIGS.
5A-5D). Morphological analysis by Giemsa staining indicated that a
percentage of CD34.sup.+ expanded cells underwent full maturation
into polychromatophilic erythrocyte/erythrocytes (FIG. 5D). In a
subsequent experiment, 11.8% of HPP CD34.sup.+ cells fully
differentiated, and 2.37% of UCB CD34.sup.+ cells fully
differentiated, into polychromatophilic erythrocyte/erythrocytes as
determined by staining with TO-PRO.RTM.-3, a nuclear stain that
detects DNA.
[0172] Adherent placental stem cells usable as feeder cells can be
obtained by mechanical disruption and enzymatic digestion of
placenta as follows. Approximately 1 g tissue samples are incubated
in a shaker in 1 mg/mL collagenase 1A for 1 hour at 37.degree. C.
at 100 RPM followed by incubation with 0.25% trypsin for 30 minutes
at 37.degree. C. at 100 RPM. The digested tissue is washed three
times with culture medium prior to transferring washed cells into
75 cm.sup.2 tissue culture flasks. The stem cells are maintained in
media consisting of 60% v/v Dulbecco's Modified Eagle's Medium-Low
Glucose (DMEM-LG), 40% v/v MCDB-201, supplemented with 2% v/v fetal
calf serum, 1.times. insulin-transferrin-selenium, lx
lenolenic-acid-bovine-serum-albumin (LA-BSA), 10.sup.-9 M
dexamethasone, 10.sup.-4M ascorbic acid 2-phosphate, 10 ng/ml
epidermal growth factor, platelet derived-growth factor--BB (10
ng/ml), and 100 U penicillin/1000 U streptomycin, in a 5% CO.sub.2
and humidified incubator at 37.degree. C. Non-adherent cells are
removed 12 to 24 hours post plating and half of the medium is
exchanged every 3-4 days. Cells are subcultured using 0.25%
trypsin-EDTA and replated at about 4.times.10.sup.3 cells/cm.sup.2
for continued expansion.
6.5. Example 5: Method and Bioreactor for Generating Units of
Erythrocytes
[0173] This Example provides a method of producing erythrocytes,
and a bioreactor that enables the production of units of mature
erythrocytes. In this particular example, the bioreactor enables
the production of administrable units of erythrocytes using a
five-step process. In the first step, hematopoietic cells, e.g.,
CD34.sup.+ cells, are isolated. In the second step, the CD34.sup.+
cells are expanded using an immunomodulatory compound, e.g.,
pomalidomide. In the third step, the CD34.sup.+ cells are expanded
in the bioreactor exemplified herein, in a co-culture with adherent
placental stem cells, in conjunction with removal of
lineage-committed cells. Fourth, remaining uncommitted
hematopoietic cells are differentiated to erythrocytes. Finally, in
the fifth step, erythrocytes are isolated and collected into
administrable units.
[0174] Steps 1 and 2, the isolation and initial expansion of
hematopoietic cells, are accomplished as described in Examples 3
and 4, above.
[0175] Steps 3 and 4 are accomplished using a bioreactor. The
bioreactor comprises a hollow fiber chamber (1) seeded with
placental stem cells (2) and an element for gas provision to the
medium (3). The bioreactor further comprises a coupled cell
sorter/separator element (4) that allows for the continuous
separation of committed hematopoietic cells, fully-differentiated
erythrocytes, or both. The cell separation element can separate the
cells from the hematopoietic cells using, e.g., magnetic cell
separation or fluorescence-activated cell separation
techniques.
[0176] To initiate cell culture, approximately 5.times.10.sup.7
hematopoietic cells, e.g., CD34.sup.+ hematopoietic cells, are
inoculated into the bioreactor.
6.6. Example 6: Collection of Erythrocytes
[0177] This Example exemplifies several methods of the separation
of erythrocytes from other lineage committed cells.
[0178] Method 1: Erythrocytes, e.g., erythrocytes collected from
the cell separation element of the bioreactor exemplified herein,
and hetastarch solution are mixed 3:1 (v:v) in a Baxter collection
bag and placed in an upright position on a plasma extractor.
Erythrocytes sediment after 50 to 70 minutes. Non-sedimented cells
are forced out by the plasma extractor. Sedimented erythrocytes
left in the bag can be further collected by centrifugation at 400 g
for 10 minutes. After removing the supernatant, erythrocytes are
resuspended in an appropriate amount of desired medium.
[0179] Method 2--Immunomagnetic separation: Glycophorin A.sup.+
cells, e.g., erythrocytes collected from the cell separation
element of the bioreactor exemplified herein, are magnetically
labeled with Glycophorin A (CD235a) MicroBeads (Miltenyi Biotech).
The cell suspension is then loaded into a tube which is placed in
the magnetic field of an EASYSEP.RTM. magnet. The magnetically
labeled Glycophorin A.sup.+ cells are retained inside the tube,
while the unlabeled cells are poured off the tube. After removal of
the tube from the magnetic field, the magnetically retained
Glycophorin A.sup.+ cells can be separated from the magnetic beads
and resuspended in an appropriate amount of desired medium.
[0180] Method 3--Flow cytometry cell separation: Erythrocytes,
e.g., erythrocytes collected from the cell separation element of
the bioreactor exemplified herein, in 500 .mu.L PBS/FBS with 1
.mu.L Fc Block (1/500). 150 .mu.L of the cell suspension is added
to each well of a 96 well V-bottom dish. 50 .mu.L 1.degree. Ab
Master Mix (the mix is a 1/25 dilution of each primary Ab in
PBS/FBS) is added to the cells. One well is included with a
combination of isotype controls for setting voltage, as well as one
well for each of the primary Ab as single positive controls for
setting compensation. The cells are incubated 60 min at 4.degree.
C., then centrifuged at 1500 RPM for two minutes. The supernatant
is discarded. The wells are washed with 200 .mu.L PBS/FBS to each
well, and mixed by pipetting up and down. The cells are then
immediately spun at 1500 RPM .times.2 min; the supernatant is
discarded. 150 .mu.L of secondary Ab (i.e. Streptavidin-TC) Master
Mix is added, and incubated 30 min at 4.degree. C., followed by
centrifugation at 1500 RPM for 5 minutes. The pellet is resuspended
in 200-500 .mu.L of PBS/FBS and transferred to 5 mL flow tubes.
Cells are then separated using a flow cytometer.
[0181] Method 4: Medium comprising erythrocytes, in continuous flow
between the cell culture element and cell separation element, is
deoxygenated by reducing or turning off the supply of oxygen from
the gas provision element, and turning on a magnet in the cell
separation element. Medium is passed through the cell separation
element for a sufficient time for the magnetic field of the magnet
to collect erythrocytes to a surface in the cell separation
element. Once a predetermined number of erythrocytes are collected,
or collection has proceeded for a predetermined amount of time, the
medium is reoxygenated, releasing the erythrocytes from the
surface.
6.7. Example 7: Bioreactor for Producing Erythrocytes
[0182] This Example describes a bioreactor design that allows for
improved production of erythrocytes from hematopoietic cells. The
bioreactor comprises a culturing element that comprises hollow
fibers in which hematopoietic cells and feeder cells are cultured.
Hematopoietic cells, e.g., hematopoietic progenitor cells, are
supplied in a bag at 5.times.10.sup.5 cells/dose, where one dose
yields one unit of blood. The cells are expanded in the presence of
IMDM medium containing 50 ng/mL SCF, 3 units/mL Epo, and 50 ng/mL
IGF-1 added through a first port. Gas provision (5% CO.sub.2 in
air) occurs through a second port. The medium in which the
hematopoietic cells are cultured is supplemented with pomalidomide
at 2.7 .mu.g/mL. The cells are cultured in contact with feeder
cells (adherent placental stem cells) that have been seeded in the
hollow fiber element of the bioreactor. During culturing, gas,
medium metabolites and medium pH in the culturing element is
monitored continuously, and are replenished or exchanged using a
programmable control device as necessary. pH of the medium in the
culturing element is maintained at approximately 7 and the culture
temperature is maintained at about 37.degree. C. Lineage-committed
cells (i.e., differentiated cells) are continuously separated and
recovered from the culture medium using a cell separation element.
The bioreactor is equipped with an independent power supply to
enable operation at a remote site, e.g., a site separate from a
site at which hematopoietic cells are initially obtained.
[0183] The present disclosure, including devices, compositions and
methods, is not to be limited in scope by the specific embodiments
described herein. Indeed, various modifications in addition to
those described herein will become apparent to those skilled in the
art from the foregoing description. Such modifications are intended
to fall within the scope of the appended claims.
[0184] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
[0185] The citation of any publication is for its disclosure prior
to the filing date and should not be construed as an admission that
the present disclosure is not entitled to antedate such publication
by virtue of prior invention.
* * * * *